Renal cell populations and uses thereof

ABSTRACT

The present invention concerns enriched heterogeneous mammalian renal cell populations characterized by biomarkers, and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/438,607, filed Apr. 24, 2015, which is a National Stage Applicationof International Patent Application PCT/US13/66707 filed Oct. 24, 2013and claims priority to U.S. Provisional Patent Application Ser. No.61/718,150, entitled “Isolated Regenerative Renal Cells and UsesThereof”, filed 24 Oct. 2012, and 61/876,616, entitled “Renal CellPopulations and Uses Thereof” filed 11 Sep. 2013, each of which theentire disclosures are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present disclosure generally relates to an enriched heterogeneousmammalian kidney-derived cell population, methods of identifying thecell population, methods for their use in the preparation of aregenerative medicine therapy, and methods for treating kidney diseaseby administration of the enriched heterogeneous kidney-derived cellpopulation to a mammalian subject are provided herein.

BACKGROUND OF THE INVENTION

Collagen and gelatin-based biomaterials have been successfully employedfor a variety of tissue engineering applications (Rohanizadeh et al. JMater Sci Mater Med 2008; 19: 1173-1182; Takemoto et al. Tissue Eng PartA 2008; 14: 1629-1638; Young et al. J Control Release 2005; 109:256-274). Both of these macromolecules are characterized by excellentbiocompatibility and low antigenicity (Cenni et al. J Biomater Sci PolymEd 2000; 11: 685-699; Lee et al. Int J Pharm 2001; 221: 1-22; Waksman etal. J Immunol 1949; 63: 427-433); however, since gelatin is obtained bythe hydrolysis of collagen, it has certain advantages over the latter:(a) it is readily available and easy to use; (b) offers options relativeto molecular weight and bloom (i.e. control over physical properties);and (c) is more flexible towards chemical modification and morestraightforward to manufacture. Moreover, from a biological standpoint,gelatin maintains cytocompatibility and cell adherence propertiessimilar to collagen Engvall et al. Int J Cancer 1977; 20: 1-5; Kim etal. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108:e94-100).

Various methods have been reported for the crosslinking of thesemacromolecules for the purpose of delaying their biodegradation toprolong their in vivo residence (in tissue engineering applications) ortailoring their drug releasing capacity (when used as drug carriers).Numerous methods have been published for chemical or photochemicalcrosslinking of collagen or gelatin (Adhirajan et al. J Microencapsul2007; 24: 647-659; Chang et al. Macromol Biosci 2007; 7: 500-507;Gagnieu et al. Biomed Mater Eng 2007; 17: 9-18; Kimura et al. J BiomaterSci Polym Ed 2010; 21: 463-476; Ma et al. J Biomed Mater Res A 2004; 71:334-342; Vandelli et al. Int J Pharm 2001; 215: 175-184; Vandelli et al.J Control Release 2004; 96: 67-84). The majority of these procedures aretargeted to reduce the susceptibility of these biomaterials to enzymaticdegradation and to extend their in vivo residence time (Chang et al.supra 2007; Ma et al. supra 2004). Other crosslinking methods aretypically employed to yield gelatin or collagen-based biomaterialssuitable as slow release drug, protein or nucleic acid carriers (Kimurasupra 2010; Vandelli supra 2004; Kommareddy et al. Nanomedicine 2007; 3:32-42; Sehgal et al. Expert Opin Drug Deliv 2009; 6: 687-695; Sutter etal. J Control Release 2007; 119: 301-312). A widely used crosslinkingagent class for collagen and gelatin as well as other tissueengineering-compatible systems is the carbodiimides (Adhirajan supra2007; Olde Damink et al. Biomaterials 1996; 17: 765-773; Pieper et al.Biomaterials 2000; 21: 581-593; Cornwell et al. Clin Podiatr Med Surg2009; 26: 507-523). These molecules are known as zero-lengthcrosslinkers and act by mediating the formation of amide bonds betweencarboxyl and primary amine functionalities present on the species to becrosslinked. In addition, carbodiimides are less cytotoxic compared toother common crosslinking agent (e.g. glutaraldehyde) (Lai et al. JMater Sci Mater Med 2010; 21: 1899-1911). Glutaraldehylde is used as acrosslinker in Cultispher™ beads. Burg U.S. Pat. No. 6,991,652 describestissue engineering composites containing three-dimensional supportconstructs for cells that may be delivered to a subject.

Regenerative medicine technologies provide next-generation therapeuticoptions for chronic kidney disease (CKD). Presnell et al. WO/2010/056328and Ilagan et al. PCT/US2011/036347 describe isolated bioactive renalcells, including tubular and erythropoietin (EPO)-producing kidney cellpopulations, and methods of isolating and culturing the same, as well asmethods of treating a subject in need with the cell populations.

There is a need for therapeutic formulations that are suitable fordelivery of active agents, such as for example, bioactive cells intissue engineering and regenerative medicine applications, to subjectsin need.

The kidney is a complex organ that performs many functions to keep theblood clean and chemically balanced. In addition to removing wastes, thekidneys release three important hormones:

erythropoietin, or EPO, which stimulates the bone marrow to make redblood cells

renin, which regulates blood pressure; and

calcitriol, the active form of vitamin D, which helps maintain calciumfor bones and for normal chemical balance in the body.

To perform these functions the kidney comprises numerous different celltypes. However, not all renal cells are required for a regenerativeresponse and identifying the combination of cells useful in eliciting aregenerative response has been the subject of investigation. Thus, thereremains a need for methods that identify a heterogeneous renal cellpopulation, i.e. bioactive cells, that finds use in the therapeuticformulations disclosed herein.

SUMMARY OF THE INVENTION

Disclosed herein is a heterogeneous mammalian renal cell population frommammalian kidney tissue. Methods for the isolation and purification ofthe mammalian kidney-derived cell population are provided. A uniquepopulation of mammalian kidney-derived cells is characterized byphenotypic characteristics, for example, biomarker phenotype. Biomarkerexpression phenotype is retained after multiple passages of themammalian kidney-derived cell population in culture and is suitable foruse in the preparation of a regenerative therapy.

Described herein are a select population of human renal cell populationcharacterized by specific biomarkers and their use.

The select population of human renal cells allows the use a smallernumbers of cells that provide a regenerative stimulus. This smallernumber is advantageous because it lowers the possibility of adverseimmunological events as well as provide a regenerative stimulus. Theselected renal cell population does not require a large proportion ofstem cells to be effective as a regenerative stimulus. The selectedrenal cell population may be recovered from a diseased kidney.

In one aspect, there is provided methods for identifying and/orcharacterizing a heterogeneous renal cell population. In one embodiment,the heterogeneous renal cell population is characterized by itsphenotypic expression of biomarkers. In certain embodiments, the renalcells are identified with one or more reagents that allow detection ofthe biomarkers on/in the heterogeneous renal cell population. Detectionof the biomarkers can be carried out by any suitable method, forexample, those based on immunofluorescent microscopy, flow cytometry,fiber-optic scanning cytometry, or laser scanning cytometry. In oneembodiment, a method of identifying a heterogeneous renal cellpopulation suitable for implantation and/or eliciting a regenerativeresponse, said method comprising the steps:

Isolating cells from a mammalian kidney sample;

Exposing said isolated cells to one or more labeled detection moiety,wherein each labeled detection moiety is directed to a differentbiomarker and is labeled with a different label;

Determining the percentage of cells that express each of said biomarker.

In one embodiment, the cell population is an SRC cell population thatexpresses two or more biomarkers listed in Tables 12.2 and 12.3. In oneembodiment, the biomarkers have levels of expression as provided inTable 12.4. In an embodiment the SRC cell population have levels ofexpression for GGT-1 and CK18 greater than 18% and 80%, respectively.

In one aspect, there is provided injectable, therapeutic formulationscontaining active agents, e.g., bioactive cells. In one embodiment, theinjectable formulation comprises bioactive cells and atemperature-sensitive cell-stabilizing biomaterial. In anotherembodiment, the a temperature-sensitive cell-stabilizing biomaterialmaintains (i) a substantially solid state at about 8° C. or below and/or(ii) a substantially liquid state at ambient temperature or above. Inone other embodiment, the bioactive cells comprise renal cells, asdescribed herein. In another embodiment, the bioactive cells aresubstantially uniformly dispersed throughout the volume of thecell-stabilizing biomaterial. In other embodiments, the biomaterial hasa solid-to-liquid transitional state between about 8° C. and aboutambient temperature or above. In one embodiment, the substantially solidstate is a gel state. In another embodiment, the cell-stabilizingbiomaterial comprises a hydrogel. In one other embodiment, the hydrogelcomprises gelatin. In other embodiments, the gelatin is present in theformulation at about 0.5% to about 1% (w/v). In one embodiment, thegelatin is present in the formulation at about 0.75% (w/v). In anotherembodiment, the formulation further includes a cell viability agent. Inone other embodiment, the cell viability agent comprises an agentselected from the group consisting of an antioxidant, an oxygen carrier,an immunomodulatory factor, a cell recruitment factor, a cell attachmentfactor, an anti-inflammatory agent, an immunosuppressant, an angiogenicfactor, and a wound healing factor. In some embodiments, the cellviability agent is an antioxidant. In one embodiment, the antioxidant is6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. In anotherembodiment, the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acidis present at about 50 μM to about 150 μM. In one other embodiment, the6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid is present atabout 100 μM. In some embodiments, the cell viability agent is an oxygencarrier. In one embodiment, the oxygen carrier is a perfluorocarbon. Inother embodiments, the cell viability agent is an immunomodulatoryagent. In one embodiment, the cell viability agent is animmunosuppressant.

In another aspect, there is provided injectable, therapeuticformulations containing bioactive renal cells. In one embodiment, theformulation comprises bioactive renal cells, about 0.75% (w/v) gelatin,and about 100 μM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid,wherein the formulation has (i) a substantially solid state at about 8°C. or below, and (ii) a substantially liquid state at ambienttemperature or above. In another embodiment, the bioactive renal cellsare substantially uniformly dispersed throughout the volume of thecell-stabilizing biomaterial. In one other embodiment, the biomaterialcomprises a solid-to-liquid transitional state between about 8° C. andabout ambient temperature. In other embodiments, the substantially solidstate is a gel state. In some embodiments, the formulation furtherincludes a cell viability agent. In yet another embodiment, the cellviability agent comprises an agent selected from the group consisting ofan antioxidant, an oxygen carrier, an immunomodulatory factor, a cellrecruitment factor, a cell attachment factor, an anti-inflammatoryagent, an angiogenic factor, and a wound healing factor. In oneembodiment, the cell viability agent is an oxygen carrier. In anotherembodiment, the oxygen carrier is a perfluorocarbon. In one otherembodiment, the cell viability agent is an immunomodulatory agent. Inother embodiments, the cell viability agent is an immunosuppressant.

In one other aspect, the present disclosure provides a formulationdescribed herein that further includes biocompatible beads. In oneembodiment, the biocompatible beads comprise a biomaterial. In anotherembodiment, the beads are crosslinked. In one other embodiment, thecrosslinked beads have a reduced susceptibility to enzymatic degradationas compared to non-crosslinked biocompatible beads. In otherembodiments, the crosslinked beads are carbodiimide-crosslinked beads.In one embodiment, the carbodiimide is selected from the groupconsisting of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimidehydrochloride (EDC), DCC—N,N′-dicyclohexylcarbodiimide (DCC), andN,N′-Diisopropylcarbodiimide (DIPC). In another embodiment, thecarbodiimide is 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimidehydrochloride (EDC). In one other embodiment, the crosslinked beadscomprise a reduced number of free primary amines as compared tonon-crosslinked beads. In other embodiments, the number of free primaryamines is detectable spectrophotometrically at about 355 nm. In someembodiments, the beads are seeded with the bioactive cells. In oneembodiment, the bioactive cells are renal cells. In another embodiment,the formulation further comprises additional biocompatible beads thatcomprise a temperature-sensitive biomaterial that maintains (i) asubstantially solid state at ambient temperature or below, and (ii) asubstantially liquid state at about 37° C. or above. In one otherembodiment, the biomaterial of the beads comprises a solid-to-liquidtransitional state between ambient temperature and about 37° C. In otherembodiments, the substantially solid state is a gel state. In oneembodiment, the biomaterial of the beads comprises a hydrogel. Inanother embodiment, the hydrogel comprises gelatin. In one otherembodiment, the beads comprise gelatin at about 5% (w/v) to about 10%(w/v). In some embodiments, the additional biocompatible beads arespacer beads. In other embodiments, the spacer beads are not seeded withbioactive cells.

In another aspect, the formulations of the present disclosure containproducts secreted by a renal cell population. In one embodiment, theformulations comprise products secreted by a renal cell populationand/or bioactive cells. In one other embodiment, the bioactive cells arerenal cells. In another embodiment, the products comprise one or more ofparacrine factors, endocrine factors, and juxtacrine factors. In oneother embodiment, the products comprise vesicles. In other embodiments,the vesicles comprise microvesicles. In one embodiment, the vesiclescomprise exosomes. In another embodiment, the vesicles comprise asecreted product selected from the group consisting of paracrinefactors, endocrine factors, juxtacrine factors, and RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of the overall NKA manufacturing processdescribed herein.

FIG. 2 A-D is a flow diagram providing further details of the processdepicted in FIG. 1 and described herein.

FIG. 3 is a graph that exemplifies the variations in culture durationand cell yields from six patients.

FIG. 4 is a picture of the SRC banding in a 7% OptiPrep® densitygradient. Reference is made to Example 5.

FIG. 5 is a bar graph depicting the expression of renal cell markers inhuman SRC populations. Reference is made to Example 12.

FIG. 6 is a bar graph depicting the enzymatic activity of human SRC.Reference is made to Example 12.

FIG. 7A-D depicts NKA injection in the kidney: (a) needle inserted intothe kidney cortex, (b) NKA delivery, (c) multiple delivery points in thekidney, and (d) final implant of the NKA (exemplary).

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are therapeutic formulations for active agents, such asbioactive cells, as well as methods of preparing the same and methods oftreating a subject in need with the formulations. The bioactive cellformulations may be suitable for heterogenous mixtures or fractions ofbioactive renal cells (BRCs). The bioactive renal cells may be isolatedrenal cells including tubular and erythropoietin (EPO)-producing kidneycells. The BRC cell populations may include enriched tubular andEPO-producing cell populations. The BRCs may be derived from or arethemselves renal cell fractions from healthy individuals. In addition,there is provided renal cell fractions obtained from an unhealthyindividual that may lack certain cellular components when compared tothe corresponding renal cell fractions of a healthy individual, yetstill retain therapeutic properties. The present disclosure alsoprovides therapeutically-active cell populations lacking cellularcomponents compared to a healthy individual, which cell populations canbe, in one embodiment, isolated and expanded from autologous sources invarious disease states.

Although bioactive cell formulations are described herein, the presentdisclosure contemplates formulations containing a variety of otheractive agents. Other suitable active agents include, without limitation,cellular aggregates, acellular biomaterials, secreted products frombioactive cells, large and small molecule therapeutics, as well ascombinations thereof. For example, one type of bioactive cells may becombined with biomaterial-based microcarriers with or withouttherapeutic molecules or another type of bioactive cells, unattachedcells may be combined with acellular particles.

1. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Principles of TissueEngineering, 3^(rd) Ed. (Edited by R Lanza, R Langer, & J Vacanti), 2007provides one skilled in the art with a general guide to many of theterms used in the present application. One skilled in the art willrecognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. Indeed, the present invention is in no way limited to themethods and materials described.

The term “cell population” as used herein refers to a number of cellsobtained by isolation directly from a suitable tissue source, usuallyfrom a mammal. The isolated cell population may be subsequently culturedin vitro. Those of ordinary skill in the art will appreciate thatvarious methods for isolating and culturing cell populations for usewith the present invention and various numbers of cells in a cellpopulation that are suitable for use in the present invention. A cellpopulation may be an unfractionated, heterogeneous cell populationderived from an organ or tissue, e.g., the kidney. For example, aheterogeneous cell population may be isolated from a tissue biopsy orfrom whole organ tissue. Alternatively, the heterogeneous cellpopulation may be derived from in vitro cultures of mammalian cells,established from tissue biopsies or whole organ tissue. Anunfractionated heterogeneous cell population may also be referred to asa non-enriched cell population. In one embodiment, the cell populationscontain bioactive cells.

The term “native organ” shall mean the organ of a living subject. Thesubject may be healthy or un-healthy. An unhealthy subject may have adisease associated with that particular organ.

The term “native kidney” shall mean the kidney of a living subject. Thesubject may be healthy or un-healthy. An unhealthy subject may have akidney disease.

The term “regenerative effect” shall mean an effect which provides abenefit to a native organ, such as the kidney. The effect may include,without limitation, a reduction in the degree of injury to a nativeorgan or an improvement in, restoration of, or stabilization of a nativeorgan function. Renal injury may be in the form of fibrosis,inflammation, glomerular hypertrophy, etc. and related to a diseaseassociated with the native organ in the subject.

The term “admixture” as used herein refers to a combination of two ormore isolated, enriched cell populations derived from an unfractionated,heterogeneous cell population. According to certain embodiments, thecell populations are renal cell populations.

An “enriched” cell population or preparation refers to a cell populationderived from a starting organ cell population (e.g., an unfractionated,heterogeneous cell population) that contains a greater percentage of aspecific cell type than the percentage of that cell type in the startingpopulation. For example, a starting kidney cell population can beenriched for a first, a second, a third, a fourth, a fifth, and so on,cell population of interest. As used herein, the terms “cellpopulation”, “cell preparation” and “cell prototype” are usedinterchangeably.

In one aspect, the term “enriched” cell population as used herein refersto a cell population derived from a starting organ cell population(e.g., a cell suspension from a kidney biopsy or cultured mammaliankidney cells) that contains a percentage of cells capable of producingEPO that is greater than the percentage of cells capable of producingEPO in the starting population. For example, the term “B4” is a cellpopulation derived from a starting kidney cell population that containsa greater percentage of EPO-producing cells, glomerular cells, andvascular cells as compared to the starting population. The cellpopulations may be enriched for one or more cell types and depleted ofone or more other cell types. For example, an enriched EPO-producingcell population may be enriched for interstitial fibroblasts anddepleted of tubular cells and collecting duct epithelial cells relativeto the interstitial fibroblasts and tubular cells in a non-enriched cellpopulation, i.e. the starting cell population from which the enrichedcell population is derived. In all embodiments citing EPO-enriched or“B4” populations, the enriched cell populations are heterogeneouspopulations of cells containing cells that can produce EPO in anoxygen-regulated manner, as demonstrated by oxygen-tunable EPOexpression from the endogenous native EPO gene.

In another aspect, an enriched renal cell population, which contains agreater percentage of a specific cell type, e.g., vascular, glomerular,or endocrine cells, than the percentage of that cell type in thestarting population, may also lack or be deficient in one or morespecific cell types, e.g., vascular, glomerular, or endocrine cells, ascompared to a starting kidney cell population derived from a healthyindividual or subject. For example, the term “B4′,” or B4 prime,” in oneaspect, is a cell population derived from a starting kidney cellpopulation that lacks or is deficient in one or more cell types, e.g.,vascular, glomerular or endocrine, depending on the disease state of thestarting specimen, as compared to a healthy individual. In oneembodiment, the B4′ cell population is derived from a subject havingchronic kidney disease. In one embodiment, the B4′ cell population isderived from a subject having focal segmental glomerulosclerosis (FSGS).In another embodiment, the B4′ cell population is derived from a subjecthaving autoimmune glomerulonephritis. In another aspect, B4′ is a cellpopulation derived from a starting cell population including all celltypes, e.g., vascular, glomerular, or endocrine cells, which is laterdepleted of or made deficient in one or more cell types, e.g., vascular,glomerular, or endocrine cells. In yet another aspect, B4′ is a cellpopulation derived from a starting cell population including all celltypes, e.g., vascular, glomerular, or endocrine cells, in which one ormore specific cell types e.g., vascular, glomerular, or endocrine cells,is later enriched. For example, in one embodiment, a B4′ cell populationmay be enriched for vascular cells but depleted of glomerular and/orendocrine cells. In another embodiment, a B4′ cell population may beenriched for glomerular cells but depleted of vascular and/or endocrinecells. In another embodiment, a B4′ cell population may be enriched forendocrine cells but depleted of vascular and/or glomerular cells. Inanother embodiment, a B4′ cell population may be enriched for vascularand endocrine cells but depleted of glomerular cells. In preferredembodiments, the B4′ cell population, alone or admixed with anotherenriched cell population, e.g., B2 and/or 83, retains therapeuticproperties. A B4′ cell population, for example, is described herein inthe Examples, e.g., Examples 11-13.

In another aspect, an enriched cell population may also refer to a cellpopulation derived from a starting kidney cell population as discussedabove that contains a percentage of cells expressing one or morevascular, glomerular and proximal tubular markers with someEPO-producing cells that is greater than the percentage of cellsexpressing one or more vascular, glomerular and proximal tubular markerswith some EPO-producing cells in the starting population. For example,the term “B3” refers to a cell population derived from a starting kidneycell population that contains a greater percentage of proximal tubularcells as well as vascular and glomerular cells as compared to thestarting population. In one embodiment, the B3 cell population containsa greater percentage of proximal tubular cells as compared to thestarting population but a lesser percentage of proximal tubular cells ascompared to the B2 cell population. In another embodiment, the B3 cellpopulation contains a greater percentage of vascular and glomerularcells markers with some EPO-producing cells as compared to the startingpopulation but a lesser percentage of vascular and glomerular cellsmarkers with some EPO-producing cells as compared to the B4 cellpopulation.

In another aspect, an enriched cell population may also refer to a cellpopulation derived from a starting kidney cell population as discussedabove that contains a percentage of cells expressing one or more tubularcell markers that is greater than the percentage of cells expressing oneor more tubular cell markers in the starting population. For example,the term “B2” refers to a cell population derived from a starting kidneycell population that contains a greater percentage of tubular cells ascompared to the starting population. In addition, a cell populationenriched for cells that express one or more tubular cell markers (or“B2”) may contain some epithelial cells from the collecting duct system.Although the cell population enriched for cells that express one or moretubular cell markers (or “B2”) is relatively depleted of EPO-producingcells, glomerular cells, and vascular cells, the enriched population maycontain a smaller percentage of these cells (EPO-producing, glomerular,and vascular) in comparison to the starting population. In general, aheterogeneous cell population is depleted of one or more cell types suchthat the depleted cell population contains a lesser proportion of thecell type(s) relative to the proportion of the cell type(s) contained inthe heterogeneous cell population prior to depletion. The cell typesthat may be depleted are any type of kidney cell. For example, incertain embodiments, the cell types that may be depleted include cellswith large granularity of the collecting duct and tubular system havinga density of <about 1.045 g/ml, referred to as “B1”. In certain otherembodiments, the cell types that may be depleted include debris andsmall cells of low granularity and viability having a density of >about1.095 g/ml, referred to as “B5”. In some embodiments, the cellpopulation enriched for tubular cells is relatively depleted of all ofthe following: “B1”, “B5”, oxygen-tunable EPO-expressing cells,glomerular cells, and vascular cells.

The term “hypoxic” culture conditions as used herein refers to cultureconditions in which cells are subjected to a reduction in availableoxygen levels in the culture system relative to standard cultureconditions in which cells are cultured at atmospheric oxygen levels(about 21%). Non-hypoxic conditions are referred to herein as normal ornormoxic culture conditions.

The term “oxygen-tunable” as used herein refers to the ability of cellsto modulate gene expression (up or down) based on the amount of oxygenavailable to the cells. “Hypoxia-inducible” refers to the upregulationof gene expression in response to a reduction in oxygen tension(regardless of the pre-induction or starting oxygen tension).

The term “biomaterial” as used herein refers to a natural or syntheticbiocompatible material that is suitable for introduction into livingtissue. A natural biomaterial is a material that is made by ororiginates from a living system. Synthetic biomaterials are materialswhich are not made by or do not originate from a living system. Thebiomaterials disclosed herein may be a combination of natural andsynthetic biocompatible materials. As used herein, biomaterials include,for example, polymeric matrices and scaffolds. Those of ordinary skillin the art will appreciate that the biomaterial(s) may be configured invarious forms, for example, as porous foam, gels, liquids, beads,solids, and may comprise one or more natural or synthetic biocompatiblematerials. In one embodiment, the biomaterial is the liquid form of asolution that is capable of becoming a hydrogel.

The term “modified release” or the equivalent terms “controlledrelease”, “delayed release”, or “slow release” refer to formulationsthat release an active agent, such as bioactive cells, over time or atmore than one point in time following administration to an individual.Modified release of an active agent, which can occur over a range ofdesired times, e.g., minutes, hours, days, weeks, or longer, dependingupon the formulation, is in contrast to standard formulations in whichsubstantially the entire dosage unit is available immediately afteradministration. For tissue engineering and regenerative medicineapplications, preferred modified release formulations provide for therelease of an active agent at multiple time points following localadministration (e.g., administration of an active agent directly to asolid organ). For example, a modified release formulation of bioactivecells would provide an initial release of cells immediately at the timeof administration and a later, second release of cells at a later time.The time delay for the second release of an active agent may be minutes,hours, or days after the initial administration. In general, the periodof time for delay of release corresponds to the period of time that ittakes for a biomaterial carrier of the active agent to lose itstructural integrity. The delayed release of an active agent begins assuch integrity begins to degrade and is completed by the time integrityfails completely. Those of ordinary skill in the art will appreciateother suitable mechanisms of release.

The term “anemia” as used herein refers to a deficit in red blood cellnumber and/or hemoglobin levels due to inadequate production offunctional EPO protein by the EPO-producing cells of a subject, and/orinadequate release of EPO protein into systemic circulation, and/or theinability of erythroblasts in the bone marrow to respond to EPO protein.A subject with anemia is unable to maintain erythroid homeostasis. Ingeneral, anemia can occur with a decline or loss of kidney function(e.g., chronic renal failure), anemia associated with relative EPOdeficiency, anemia associated with congestive heart failure, anemiaassociated with myelo-suppressive therapy such as chemotherapy oranti-viral therapy (e.g., AZT), anemia associated with non-myeloidcancers, anemia associated with viral infections such as HIV, and anemiaof chronic diseases such as autoimmune diseases (e.g., rheumatoidarthritis), liver disease, and multi-organ system failure.

The term “EPO-deficiency” refers to any condition or disorder that istreatable with an erythropoietin receptor agonist (e.g., recombinant EPOor EPO analogs), including anemia.

The term “organ-related disease” as used herein refers to disordersassociated with any stage or degree of acute or chronic organ failurethat results in a loss of the organ's ability to perform its function.

The term “kidney disease” as used herein refers to disorders associatedwith any stage or degree of acute or chronic renal failure that resultsin a loss of the kidney's ability to perform the function of bloodfiltration and elimination of excess fluid, electrolytes, and wastesfrom the blood. Kidney disease also includes endocrine dysfunctions suchas anemia (erythropoietin-deficiency), and mineral imbalance (Vitamin Ddeficiency). Kidney disease may originate in the kidney or may besecondary to a variety of conditions, including (but not limited to)heart failure, hypertension, diabetes, autoimmune disease, or liverdisease. Kidney disease may be a condition of chronic renal failure thatdevelops after an acute injury to the kidney. For example, injury to thekidney by ischemia and/or exposure to toxicants may cause acute renalfailure; incomplete recovery after acute kidney injury may lead to thedevelopment of chronic renal failure.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures for kidney disease, anemia, EPOdeficiency, tubular transport deficiency, or glomerular filtrationdeficiency wherein the object is to reverse, prevent or slow down(lessen) the targeted disorder. Those in need of treatment include thosealready having a kidney disease, anemia, EPO deficiency, tubulartransport deficiency, or glomerular filtration deficiency as well asthose prone to having a kidney disease, anemia, EPO deficiency, tubulartransport deficiency, or glomerular filtration deficiency or those inwhom the kidney disease, anemia, EPO deficiency, tubular transportdeficiency, or glomerular filtration deficiency is to be prevented. Theterm “treatment” as used herein includes the stabilization and/orimprovement of kidney function.

The term “in vivo contacting” as used herein refers to direct contact invivo between products secreted by an enriched population of cells and anative organ. For example, products secreted by an enriched populationof renal cells (or an admixture or construct containing renalcells/renal cell fractions) may in vivo contact a native kidney. Thedirect in vivo contacting may be paracrine, endocrine, or juxtacrine innature. The products secreted may be a heterogeneous population ofdifferent products described herein.

The term “ribonucleic acid” or “RNA” as used herein refers to a chain ofnucleotide units where each unit is made up of a nitrogenous base, aribose sugar, and a phosphate. The RNA may be in single or doublestranded form. The RNA may be part of, within, or associated with avesicle. The vesicle may be an exosome. RNA includes, withoutlimitation, mRNAs, rRNA, small RNAs, snRNAs, snoRNAs, microRNAs(miRNAs), small interfering RNAs (siRNAs), and noncoding RNAs. The RNAis preferably human RNA.

The term “construct” refers to one or more cell populations deposited onor in a surface of a scaffold or matrix made up of one or more syntheticor naturally-occurring biocompatible materials. The one or more cellpopulations may be coated with, deposited on, embedded in, attached to,seeded, or entrapped in a biomaterial made up of one or more syntheticor naturally-occurring biocompatible biomaterials, polymers, proteins,or peptides. The one or more cell populations may be combined with abiomaterial or scaffold or matrix in vitro or in vivo. In general, theone or more biocompatible materials used to form thescaffold/biomaterial is selected to direct, facilitate, or permit theformation of multicellular, three-dimensional, organization of at leastone of the cell populations deposited thereon. The one or morebiomaterials used to generate the construct may also be selected todirect, facilitate, or permit dispersion and/or integration of theconstruct or cellular components of the construct with the endogenoushost tissue, or to direct, facilitate, or permit the survival,engraftment, tolerance, or functional performance of the construct orcellular components of the construct.

The term “marker” or “biomarker” refers generally to a DNA, RNA,protein, carbohydrate, or glycolipid-based molecular marker, theexpression or presence of which in a cultured cell population can bedetected by standard methods (or methods disclosed herein) and isconsistent with one or more cells in the cultured cell population beinga particular type of cell. Such biomarkers include, but are not limitedto, the genes set forth in Tables X and Y. The marker may be apolypeptide expressed by the cell or an identifiable physical locationon a chromosome, such as a gene, a restriction endonuclease recognitionsite or a nucleic acid encoding a polypeptide (e.g., an mRNA) expressedby the native cell. The marker may be an expressed region of a genereferred to as a “gene expression marker”, or some segment of DNA withno known coding function. The biomarkers may be cell-derived, e.g.,secreted, products.

The terms “biomarker signature,” “signature,” “biomarker expressionsignature,” or “expression signature” are used interchangeably hereinand refer to one or a combination of biomarkers whose expression is anindicator of the cell type(s), e.g., epithelial, tubular, etc.comprising a cell population, e.g., bioactive renal cells. The biomarkersignature may serve as an indictor of suitability of the cell populationfor use in the methods and manufactures provided for herein. In someembodiments, the biomarker signature is a “gene signature.” The term“gene signature” is used interchangeably with “gene expressionsignature” and refers to one or a combination of polynucleotides whoseexpression is an indicator of cell type, e.g., epithelial, tubular, etc.In some embodiments, the biomarker signature is a “protein signature.”The term “protein signature” is used interchangeably with “proteinexpression signature” and refers to one or a combination of polypeptideswhose expression is an indicator of cell type, e.g., epithelial,tubular, etc.

The terms “level of expression” or “expression level” are usedinterchangeably and generally refer to either the amount of apolynucleotide or an amino acid product or protein in a biologicalsample, or the percentage of cells expressing the polynucleotide or anamino acid product or protein. “Expressing” or “Expression” andgrammatical variants thereof refer to the presence of a polynucleotideor an amino acid product or protein in a detectable amount in abiological sample. For example, a protein that is detectable (abovebackground or control values) may be said to express the protein.Similarly, if a portion of cells in a sample express the protein thesample may be said to express the protein. In the alternative, thesample may be said to have a level of expression relating to thepercentage of cells expressing the protein, e.g., if 60% of the cells ina sample express the protein then the level of expression is 60%.

The terms “differentially expressed gene,” “differential geneexpression” and their synonyms, which are used interchangeably, refer toa gene whose expression is activated to a higher or lower level in afirst cell or cell population, relative to its expression in a secondcell or cell population. The terms also include genes whose expressionis activated to a higher or lower level at different stages over timeduring passage of the first or second cell in culture. It is alsounderstood that a differentially expressed gene may be either activatedor inhibited at the nucleic acid level or protein level, or may besubject to alternative splicing to result in a different polypeptideproduct. Such differences may be evidenced by a change in mRNA levels,surface expression, secretion or other partitioning of a polypeptide,for example. Differential gene expression may include a comparison ofexpression between two or more genes or their gene products, or acomparison of the ratios of the expression between two or more genes ortheir gene products, or even a comparison of two differently processedproducts of the same gene, which differ between the first cell and thesecond cell. Differential expression includes both quantitative, as wellas qualitative, differences in the temporal or cellular expressionpattern in a gene or its expression products among, for example, thefirst cell and the second cell. For the purpose of this disclosure,“differential gene expression” is considered to be present when there isa difference between the expression of a given gene in the first celland the second cell. The differential expression of a marker may be incells from a patient before administration of a cell population,admixture, or construct (the first cell) relative to expression in cellsfrom the patient after administration (the second cell).

The terms “inhibit”, “down-regulate”, “under-express” and “reduce” areused interchangeably and mean that the expression of a gene, or level ofRNA molecules or equivalent RNA molecules encoding one or more proteinsor protein subunits, or activity of one or more proteins or proteinsubunits, is reduced relative to one or more controls, such as, forexample, one or more positive and/or negative controls. Theunder-expression may be in cells from a patient before administration ofa cell population, admixture, or construct relative to cells from thepatient after administration.

The term “up-regulate” or “over-express” is used to mean that theexpression of a gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is elevated relative to oneor more controls, such as, for example, one or more positive and/ornegative controls. The over-expression may be in cells from a patientafter administration of a cell population, admixture, or constructrelative to cells from the patient before administration.

The term “subject” shall mean any single human subject, including apatient, eligible for treatment, who is experiencing or has experiencedone or more signs, symptoms, or other indicators of an organ-relateddisease, such as kidney disease, anemia, or EPO deficiency. Suchsubjects include without limitation subjects who are newly diagnosed orpreviously diagnosed and are now experiencing a recurrence or relapse,or are at risk for a kidney disease, anemia, or EPO deficiency, nomatter the cause. The subject may have been previously treated for akidney disease, anemia, or EPO deficiency, or not so treated.

The term “patient” refers to any single animal, more preferably a mammal(including such non-human animals as, for example, dogs, cats, horses,rabbits, zoo animals, cows, pigs, sheep, and non-human primates) forwhich treatment is desired. Most preferably, the patient herein is ahuman.

The term “sample” or “patient sample” or “biological sample” shallgenerally mean any biological sample obtained from a subject or patient,body fluid, body tissue, cell line, tissue culture, or other source. Theterm includes tissue biopsies such as, for example, kidney biopsies. Theterm includes cultured cells such as, for example, cultured mammaliankidney cells. Methods for obtaining tissue biopsies and cultured cellsfrom mammals are well known in the art. If the term “sample” is usedalone, it shall still mean that the “sample” is a “biological sample” or“patient sample”, i.e., the terms are used interchangeably.

The term “test sample” refers to a sample from a subject that has beentreated by a method disclosed herein. The test sample may originate fromvarious sources in the mammalian subject including, without limitation,blood, semen, serum, urine, bone marrow, mucosa, tissue, etc.

The term “control” or “control sample” refers a negative or positivecontrol in which a negative or positive result is expected to helpcorrelate a result in the test sample. Controls that are suitableinclude, without limitation, a sample known to exhibit indicatorscharacteristic of normal erythroid homeostasis, a sample known toexhibit indicators characteristic of anemia, a sample obtained from asubject known not to be anemic, and a sample obtained from a subjectknown to be anemic. Additional controls suitable for use in the methodsprovided herein include, without limitation, samples derived fromsubjects that have been treated with pharmacological agents known tomodulate erythropoiesis (e.g., recombinant EPO or EPO analogs). Inaddition, the control may be a sample obtained from a subject prior tobeing treated by a method disclosed herein. An additional suitablecontrol may be a test sample obtained from a subject known to have anytype or stage of kidney disease, and a sample from a subject known notto have any type or stage of kidney disease. A control may be a normalhealthy matched control. Those of skill in the art will appreciate othercontrols suitable for use herein.

“Regeneration prognosis”, “regenerative prognosis”, or “prognostic forregeneration” generally refers to a forecast or prediction of theprobable regenerative course or outcome of the administration orimplantation of a cell population, admixture or construct describedherein. For a regeneration prognosis, the forecast or prediction may beinformed by one or more of the following: improvement of a functionalorgan (e.g., the kidney) after implantation or administration,development of a functional kidney after implantation or administration,development of improved kidney function or capacity after implantationor administration, and expression of certain markers by the nativekidney following implantation or administration.

“Regenerated organ” refers to a native organ after implantation oradministration of a cell population, admixture, or construct asdescribed herein. The regenerated organ is characterized by variousindicators including, without limitation, development of function orcapacity in the native organ, improvement of function or capacity in thenative organ, and the expression of certain markers in the native organ.Those of ordinary skill in the art will appreciate that other indicatorsmay be suitable for characterizing a regenerated organ.

“Regenerated kidney” refers to a native kidney after implantation oradministration of a cell population, admixture, or construct asdescribed herein. The regenerated kidney is characterized by variousindicators including, without limitation, development of function orcapacity in the native kidney, improvement of function or capacity inthe native kidney, and the expression of certain markers in the nativekidney. Those of ordinary skill in the art will appreciate that otherindicators may be suitable for characterizing a regenerated kidney.

The term “cellular aggregate” or “spheroid” refers to an aggregate orassembly of cells cultured to allow 3D growth as opposed to growth as amonolayer. It is noted that the term “spheroid” does not imply that theaggregate is a geometric sphere. The aggregate may be highly organizedwith a well defined morphology or it may be an unorganized mass; it mayinclude a single cell type or more than one cell type. The cells may beprimary isolates, or a permanent cell line, or a combination of the two.Included in this definition are organoids and organotypic cultures.

The term “ambient temperature” refers to the temperature at which theformulations of the present disclosure will be administered to asubject. Generally, the ambient temperature is the temperature of atemperature-controlled environment. Ambient temperature ranges fromabout 18° C. to about 30° C. In one embodiment, ambient temperature isabout 18° C., about 19° C., about 20° C., about 21° C., about 22° C.,about 23° C., about 24° C., about 25° C., about 26° C., about 27° C.,about 28° C., about 29° C., or about 30° C.

The word “label” when used herein refers to a compound or compositionthat is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable. The term isintended to encompass direct labeling of a probe or antibody by coupling(i.e., physically linking) a detectable substance to the probe orantibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin.

The term “detection” includes any means of detecting, including directand indirect detection.

A “kit” is any manufacture (e.g., a package or container) comprising atleast one reagent, e.g., a medicament for treatment of an kidneydisease, or a probe for specifically detecting a biomarker gene orprotein as disclosed herein. The manufacture is preferably promoted,distributed, or sold as a unit for performing the methods disclosedherein.

2. Cell Populations

The formulations of the present disclosure may contain isolated,heterogeneous populations of kidney cells, and admixtures thereof,enriched for specific bioactive components or cell types and/or depletedof specific inactive or undesired components or cell types for use inthe treatment of kidney disease, i.e., providing stabilization and/orimprovement and/or regeneration of kidney function, were previouslydescribed in Presnell et al. U.S. 2011-0117162 and Ilagan et al.PCT/US2011/036347, the entire contents of which are incorporated hereinby reference. The formulations may contain isolated renal cell fractionsthat lack cellular components as compared to a healthy individual yetretain therapeutic properties, i.e., provide stabilization and/orimprovement and/or regeneration of kidney function. The cellpopulations, cell fractions, and/or admixtures of cells described hereinmay be derived from healthy individuals, individuals with a kidneydisease, or subjects as described herein.

The present disclosure provides formulations which are suitable for usewith various bioactive cell populations including, without limitation,isolated cell population(s), cell fraction(s), admixture(s), enrichedcell population(s), cellular aggregate(s), and any combination thereof.In an embodiment, the bioactive cell populations are bioactive renalcells.

Bioactive Cell Populations

The present disclosure contemplates therapeutic formulations suitablefor bioactive cell populations that are to be administered to targetorgans or tissue in a subject in need. A bioactive cell populationgenerally refers to a cell population potentially having therapeuticproperties upon administration to a subject. For example, uponadministration to a subject in need, a bioactive renal cell populationcan provide stabilization and/or improvement and/or regeneration ofkidney function in the subject. The therapeutic properties may include aregenerative effect.

Bioactive cell populations include, without limitation, stem cells(e.g., pluripotent, multipotent, oligopotent, or unipotent) such asembryonic stem cells, amniotic stem cells, adult stem cells (e.g.,hematopoietic, mammary, intestinal, mesenchymal, placental, lung, bonemarrow, blood, umbilical cord, endothelial, dental pulp, adipose,neural, olfactory, neural crest, testicular), induced pluripotent stemcells; genetically modified cells; as well as cell populations or tissueexplants derived from any source of the body. The formulations of thepresent disclosure may also be used with renal adipose-derived cellpopulations as described in Basu et al. PCT/US11/39859 filed on Jun. 9,2011; and with the adipose-derived or peripheral blood-derived smoothmuscle cells described in Ludlow et al. U.S. 2010-0131075 and Ludlow etal. PCT/US11/35058 filed on May 3, 2011; or bladder-derived urothelialor smooth muscle cells as described in Atala U.S. Pat. No. 6,576,019,each of which is incorporate herein by reference in its entirety. Thebioactive cell populations may be isolated, enriched, purified,homogeneous, or heterogeneous in nature. Those of ordinary skill in theart will appreciate other bioactive cell populations that are suitablefor use in the formulations of the present disclosure.

In one embodiment, the source of cells is the same as the intendedtarget organ or tissue. For example, renal cells may be sourced from thekidney to be used in a formulation to be administered to the kidney. Inanother embodiment, the source of cells is not the same as the intendedtarget organ or tissue. For example, erythropoietin-expressing cells maybe sourced from renal adipose to be used in a formulation to beadministered to the kidney.

In one aspect, the present disclosure provides formulations containingcertain subfractions of a heterogeneous population of renal cells,enriched for bioactive components and depleted of inactive or undesiredcomponents provide superior therapeutic and regenerative outcomes thanthe starting population. For example, bioactive renal cells describedherein, e.g., B2, B4, and B3, which are depleted of inactive orundesired components, e.g., B1 and B5, alone or admixed, can be part ofa formulation to be used for the stabilization and/or improvement and/orregeneration of kidney function.

In another aspect, the formulations contain a specific subfraction, B4,depleted of or deficient in one or more cell types, e.g., vascular,endocrine, or endothelial, i.e., B4′, that retain therapeuticproperties, e.g., stabilization and/or improvement and/or regenerationof kidney function, alone or when admixed with other bioactivesubfractions, e.g., B2 and/or B3. In a preferred embodiment, thebioactive cell population is B2. In certain embodiments, the B2 cellpopulation is admixed with B4 or B4′. In other embodiments, the B2 cellpopulation is admixed with B3. In other embodiments, the B2 cellpopulation is admixed with both B3 and B4, or specific cellularcomponents of B3 and/or B4.

The B2 cell population is characterized by expression of a tubular cellmarker selected from the group consisting of one or more of thefollowing: megalin, cubilin, hyaluronic acid synthase 2 (HAS2), VitaminD3 25-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad),Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogenefamily (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containingion transport regulator 4 (Fxyd4), solute carrier family 9(sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3(Aldh1a3), and Calpain-8 (Capn8), and collecting duct marker Aquaporin-4(Aqp4). B2 is larger and more granulated than B3 and/or B4 and thushaving a buoyant density between about 1.045 g/ml and about 1.063 g/ml(rodent), between about 1.045 g/ml and 1.052 g/ml (human), and betweenabout 1.045 g/ml and about 1.058 g/ml (canine).

The B3 cell population is characterized by the expression of vascular,glomerular and proximal tubular markers with some EPO-producing cells,being of an intermediate size and granularity in comparison to B2 andB4, and thus having a buoyant density between about 1.063 g/ml and about1.073 g/ml (rodent), between about 1.052 g/ml and about 1.063 g/ml(human), and between about 1.058 g/ml and about 1.063 g/ml (canine). B3is characterized by expression of markers selected from the groupconsisting of one or more of the following: aquaporin 7 (Aqp7), FXYDdomain-containing ion transport regulator 2 (Fxyd2), solute carrierfamily 17 (sodium phosphate), member 3 (Slc17a3), solute carrier family3, member 1 (Slc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase(Napsa), solute carrier family 2 (facilitated glucose transporter),member 2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep),transmembrane protein 27 (Tmem27), acyl-CoA synthetase medium-chainfamily member 2 (Acsm2), glutathione peroxidase 3 (Gpx3),fructose-1,6-biphosphatase 1 (Fbp1), and alanine-glyoxylateaminotransferase 2 (Agxt2). B3 is also characterized by the vascularexpression marker Platelet endothelial cell adhesion molecule (Pecam)and the glomerular expression marker podocin (Podn).

The B4 cell population is characterized by the expression of a vascularmarker set containing one or more of the following: PECAM, VEGF, KDR,HIF1a, CD31, CD146; a glomerular marker set containing one or more ofthe following: Podocin (Podn), and Nephrin (Neph); and an oxygen-tunableEPO enriched population compared to unfractionated (UNFX), 82 and B3. B4is also characterized by the expression of one or more of the followingmarkers: chemokine (C-X-C motif) receptor 4 (Cxcr4), endothelin receptortype B (Ednrb), collagen, type V, alpha 2 (Col5a2), Cadherin 5 (Cdh5),plasminogen activator, tissue (Plat), angiopoietin 2 (Angpt2), kinaseinsert domain protein receptor (Kdr), secreted protein, acidic,cysteine-rich (osteonectin) (Sparc), serglycin (Srgn), TIMPmetallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1), wingless-typeMMTV integration site family, member 4 (Wnt4), regulator of G-proteinsignaling 4 (Rgs4), Platelet endothelial cell adhesion molecule (Pecam),and Erythropoietin (Epo). B4 is also characterized by smaller, lessgranulated cells compared to either B2 or B3, with a buoyant densitybetween about 1.073 g/ml and about 1.091 g/ml (rodent), between about1.063 g/ml and about 1.091 g/mL (human and canine).

The B4′ cell population is defined as having a buoyant density ofbetween 1.063 g/mL and 1.091 g/mL and expressing one or more of thefollowing markers: PECAM, vEGF, KDR, HIF1a, podocin, nephrin, EPO, CK7,CK8/18/19. In one embodiment, the B4′ cell population is characterizedby the expression of a vascular marker set containing one or more of thefollowing: PECAM, vEGF, KDR, HIF1a, CD31, CD146. In another embodiment,the B4′ cell population is characterized by the expression of anendocrine marker EPO. In one embodiment, the B4′ cell population ischaracterized by the expression of a glomerular marker set containingone or more of the following: Podocin (Podn), and Nephrin (Neph). Incertain embodiments, the B4′ cell population is characterized by theexpression of a vascular marker set containing one or more of thefollowing: PECAM, vEGF, KDR, HIF1a and by the expression of an endocrinemarker EPO. In another embodiment, B4′ is also characterized by smaller,less granulated cells compared to either B2 or B3, with a buoyantdensity between about 1.073 g/ml and about 1.091 g/ml (rodent), betweenabout 1.063 g/ml and about 1.091 g/mL (human and canine).

In one aspect, the present disclosure provides formulations containingan isolated, enriched B4′ population of human renal cells comprising atleast one of erythropoietin (EPO)-producing cells, vascular cells, andglomerular cells having a density between 1.063 g/mL and 1.091 g/mL. Inone embodiment, the B4′ cell population is characterized by expressionof a vascular marker. In certain embodiments, the B4′ cell population isnot characterized by expression of a glomerular marker. In someembodiments, the B4′ cell population is capable of oxygen-tunableerythropoietin (EPO) expression.

In one embodiment, formulation contains the B4′ cell population but doesnot include a B2 cell population comprising tubular cells having adensity between 1.045 g/mL and 1.052 g/mL. In another embodiment, theB4′ cell population formulation does not include a B1 cell populationcomprising large granular cells of the collecting duct and tubularsystem having a density of <1.045 g/ml. In yet another embodiment, theB4′ cell population formulation does not include a B5 cell populationcomprising debris and small cells of low granularity and viability witha density >1.091 g/ml.

In one embodiment, the B4′ cell population-containing formulation doesnot include a B2 cell population comprising tubular cells having adensity between 1.045 g/mL and 1.052 g/mL; a B1 cell populationcomprising large granular cells of the collecting duct and tubularsystem having a density of <1.045 g/ml; and a B5 cell populationcomprising debris and small cells of low granularity and viability witha density >1.091 g/ml. In some embodiments, the B4′ cell population maybe derived from a subject having kidney disease.

In one aspect, the present disclosure provides formulations containingadmixtures of human renal cells comprising a first cell population, B2,comprising an isolated, enriched population of tubular cells having adensity between 1.045 g/mL and 1.052 g/mL, and a second cell population,B4′, comprising erythropoietin (EPO)-producing cells and vascular cellsbut depleted of glomerular cells having a density between about 1.063g/mL and 1.091 g/mL, wherein the admixture does not include a B1 cellpopulation comprising large granular cells of the collecting duct andtubular system having a density of <1.045 g/ml, or a B5 cell populationcomprising debris and small cells of low granularity and viability witha density >1.091 g/ml. In certain embodiment, the B4′ cell population ischaracterized by expression of a vascular marker. In one embodiment, theB4′ cell population is not characterized by expression of a glomerularmarker. In certain embodiments, B2 further comprises collecting ductepithelial cells. In one embodiment, the formulation contains anadmixture of cells that is capable of receptor-mediated albumin uptake.In another embodiment, the admixture of cells is capable ofoxygen-tunable erythropoietin (EPO) expression. In one embodiment, theadmixture contains HAS-2-expressing cells capable of producing and/orstimulating the production of high-molecular weight species ofhyaluronic acid (HA) both in vitro and in vivo. In all embodiments, thefirst and second cell populations may be derived from kidney tissue orcultured kidney cells (Basu et al. Lipids in Health and Disease, 2011,10:171).

In one embodiment, the formulation contains an admixture that is capableof providing a regenerative stimulus upon in vivo delivery. In otherembodiments, the admixture is capable of reducing the decline of,stabilizing, or improving glomerular filtration, tubular resorption,urine production, and/or endocrine function upon in vivo delivery. Inone embodiment, the B4′ cell population is derived from a subject havingkidney disease.

In one aspect, the present disclosure provides formulations containingan isolated, enriched B4′ population of human renal cells comprising atleast one of erythropoietin (EPO)-producing cells, vascular cells, andglomerular cells having a density between 1.063 g/mL and 1.091 g/mL. Inone embodiment, the B4′ cell population is characterized by expressionof a vascular marker. In certain embodiments, the B4′ cell population isnot characterized by expression of a glomerular marker. The glomerularmarker that is not expressed may be podocin. In some embodiments, theB4′ cell population is capable of oxygen-tunable erythropoletin (EPO)expression.

In one embodiment, the B4′ cell population-containing formulation doesnot include a B2 cell population comprising tubular cells having adensity between 1.045 g/mL and 1.052 g/mL. In another embodiment, theB4′ cell population formulation does not include a B1 cell populationcomprising large granular cells of the collecting duct and tubularsystem having a density of <1.045 g/ml. In yet another embodiment, theB4′ cell population formulation does not include a B5 cell populationcomprising debris and small cells of low granularity and viability witha density >1.091 g/ml.

In one embodiment, the B4′ cell population-containing formulation doesnot include a B2 cell population comprising tubular cells having adensity between 1.045 g/mL and 1.052 g/mL; a B1 cell populationcomprising large granular cells of the collecting duct and tubularsystem having a density of <1.045 g/ml; and a B5 cell populationcomprising debris and small cells of low granularity and viability witha density >1.091 g/ml. In some embodiments, the B4′ cell population maybe derived from a subject having kidney disease.

In one aspect, the present disclosure provides formulations containingan admixture of human renal cells comprising a first cell population,B2, comprising an isolated, enriched population of tubular cells havinga density between 1.045 g/mL and 1.052 g/mL, and a second cellpopulation, B4′, comprising erythropoietin (EPO)-producing cells andvascular cells but depleted of glomerular cells having a density betweenabout 1.063 g/mL and 1.091 g/mL, wherein the admixture does not includea B1 cell population comprising large granular cells of the collectingduct and tubular system having a density of <1.045 g/ml, or a B5 cellpopulation comprising debris and small cells of low granularity andviability with a density >1.091 g/ml. In certain embodiment, the B4′cell population is characterized by expression of a vascular marker. Inone embodiment, the B4′ cell population is not characterized byexpression of a glomerular marker. In certain embodiments, B2 furthercomprises collecting duct epithelial cells. In one embodiment, theadmixture of cells is capable of receptor-mediated albumin uptake. Inanother embodiment, the admixture of cells is capable of oxygen-tunableerythropoietin (EPO) expression. In one embodiment, the admixturecontains HAS-2-expressing cells capable of producing and/or stimulatingthe production of high-molecular weight species of hyaluronic acid (HA)both in vitro and in vivo. In all embodiments, the first and second cellpopulations may be derived from kidney tissue or cultured kidney cells.

In another aspect, the present disclosure provides formulationscontaining a heterogeneous renal cell population comprising acombination of cell fractions or enriched cell populations (e.g., B1,B2, B3, B4 (or B4′), and B5). In one embodiment, the combination has abuoyant density between about 1.045 g/ml and about 1.091 g/ml. In oneother embodiment, the combination has a buoyant density between lessthan about 1.045 g/ml and about 1.099 g/ml or about 1.100 g/ml. Inanother embodiment, the combination has a buoyant density as determinedby separation on a density gradient, e.g., by centrifugation. In yetanother embodiment, the combination of cell fractions contains B2, B3,and B4 (or B4′) depleted of B1 and/or B5. In some embodiments, thecombination of cell fractions contains B2, B3, B4 (or B4′), and B5 butis depleted of B1. Once depleted of B1 and/or B5, the combination may besubsequently cultured in vitro prior to the preparation of a formulationcomprising the combination of B2, B3, and B4 (or B4′) cell fractions.

The inventors of the present disclosure have surprisingly discoveredthat in vitro culturing of a B1-depleted combination of B2, B3, B4, andB5 results in depletion of B5. In one embodiment, B5 is depleted afterat least one, two, three, four, or five passages. In one otherembodiment, the B2, B3, B4, and B5 cell fraction combination that ispassaged under the conditions described herein provides a passaged cellpopulation having B5 at a percentage that is less than about 5%, lessthan about 4%, less than about 3%, less than about 2%, less than about1%, or less than about 0.5% of the passaged cell population.

In another embodiment, B4′ is part of the combination of cell fractions.In one other embodiment, the in vitro culturing depletion of B5 is underhypoxic conditions.

In one embodiment, the formulation contains an admixture that is capableof providing a regenerative stimulus upon in vivo delivery. In otherembodiments, the admixture is capable of reducing the decline of,stabilizing, or improving glomerular filtration, tubular resorption,urine production, and/or endocrine function upon in vivo delivery. Inone embodiment, the B4′ cell population is derived from a subject havingkidney disease.

In a preferred embodiment, the formulation contains an admixture thatcomprises B2 in combination with B3 and/or B4. In another preferredembodiment, the admixture comprises B2 in combination with B3 and/orB4′. In other preferred embodiments, the admixture consists of orconsists essentially of (i) B2 in combination with B3 and/or B4; or (ii)B2 in combination with B3 and/or B4′.

The admixtures that contain a B4′ cell population may contain B2 and/orB3 cell populations that are also obtained from a non-healthy subject.The non-healthy subject may be the same subject from which the B4′fraction was obtained. In contrast to the B4′ cell population, the B2and B3 cell populations obtained from non-healthy subjects are typicallynot deficient in one or more specific cell types as compared to astarting kidney cell population derived from a healthy individual.

As described in Presnell et al. WO/2010/056328, it has been found thatthe B2 and B4 cell preparations are capable of expressing highermolecular weight species of hyaluronic acid (HA) both in vitro and invivo, through the actions of hyaluronic acid synthase-2 (HAS-2)—a markerthat is enriched more specifically in the B2 cell population. Treatmentwith B2 in a 5/6 Nx model was shown to reduce fibrosis, concomitant withstrong expression HAS-2 expression in vivo and the expected productionof high-molecular-weight HA within the treated tissue. Notably, the 5/6Nx model left untreated resulted in fibrosis with limited detection ofHAS-2 and little production of high-molecular-weight HA. Without wishingto be bound by theory, it is hypothesized that this anti-inflammatoryhigh-molecular weight species of HA produced predominantly by B2 (and tosome degree by B4) acts synergistically with the cell preparations inthe reduction of renal fibrosis and in the aid of renal regeneration.Accordingly, the instant disclosure includes formulations containing thebioactive renal cells described herein along with a biomaterialcomprising hyaluronic acid. Also contemplated by the instant disclosureis the provision of a biomaterial component of the regenerative stimulusvia direct production or stimulation of production by the implantedcells.

In one aspect, the present disclosure provides formulations that containisolated, heterogeneous populations of EPO-producing kidney cells foruse in the treatment of kidney disease, anemia and/or EPO deficiency ina subject in need. In one embodiment, the cell populations are derivedfrom a kidney biopsy. In another embodiment, the cell populations arederived from whole kidney tissue. In one other embodiment, the cellpopulations are derived from in vitro cultures of mammalian kidneycells, established from kidney biopsies or whole kidney tissue. In allembodiments, these populations are unfractionated cell populations, alsoreferred to herein as non-enriched cell populations.

In another aspect, the present disclosure provides formulations thatcontain isolated populations of erythropoietin (EPO)-producing kidneycells that are further enriched such that the proportion ofEPO-producing cells in the enriched subpopulation is greater relative tothe proportion of EPO-producing cells in the starting or initial cellpopulation. In one embodiment, the enriched EPO-producing cell fractioncontains a greater proportion of interstitial fibroblasts and a lesserproportion of tubular cells relative to the interstitial fibroblasts andtubular cells contained in the unenriched initial population. In certainembodiments, the enriched EPO-producing cell fraction contains a greaterproportion of glomerular cells and vascular cells and a lesserproportion of collecting duct cells relative to the glomerular cells,vascular cells and collecting duct cells contained in the unenrichedinitial population. In such embodiments, these populations are referredto herein as the “B4” cell population.

In another aspect, the present disclosure provides formulationscontaining an EPO-producing kidney cell population that is admixed withone or more additional kidney cell populations. In one embodiment, theEPO-producing cell population is a first cell population enriched forEPO-producing cells, e.g., B4. In another embodiment, the EPO-producingcell population is a first cell population that is not enriched forEPO-producing cells, e.g., B2. In another embodiment, the first cellpopulation is admixed with a second kidney cell population. In someembodiments, the second cell population is enriched for tubular cells,which may be demonstrated by the presence of a tubular cell phenotype.In another embodiment, the tubular cell phenotype may be indicated bythe presence of one tubular cell marker. In another embodiment, thetubular cell phenotype may be indicated by the presence of one or moretubular cell markers. The tubular cell markers include, withoutlimitation, megalin, cubilin, hyaluronic acid synthase 2 (HAS2), VitaminD3 25-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad),Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogenefamily (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containingion transport regulator 4 (Fxyd4), solute carrier family 9(sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3(Aldh1a3), and Calpain-8 (Capn8). In another embodiment, the first cellpopulation is admixed with at least one of several types of kidney cellsincluding, without limitation, interstitium-derived cells, tubularcells, collecting duct-derived cells, glomerulus-derived cells, and/orcells derived from the blood or vasculature.

The formulations of the present disclosure may include EPO-producingkidney cell populations containing B4 or B4′ in the form of an admixturewith B2 and/or B3, or in the form of an enriched cell population, e.g.,B2+B3+B4/B4′.

In one aspect, the formulation contains EPO-producing kidney cellpopulations that are characterized by EPO expression andbioresponsiveness to oxygen, such that a reduction in the oxygen tensionof the culture system results in an induction in the expression of EPO.In one embodiment, the EPO-producing cell populations are enriched forEPO-producing cells. In one embodiment, the EPO expression is inducedwhen the cell population is cultured under conditions where the cellsare subjected to a reduction in available oxygen levels in the culturesystem as compared to a cell population cultured at normal atmospheric(^(˜)21%) levels of available oxygen. In one embodiment, EPO-producingcells cultured in lower oxygen conditions express greater levels of EPOrelative to EPO-producing cells cultured at normal oxygen conditions. Ingeneral, the culturing of cells at reduced levels of available oxygen(also referred to as hypoxic culture conditions) means that the level ofreduced oxygen is reduced relative to the culturing of cells at normalatmospheric levels of available oxygen (also referred to as normal ornormoxic culture conditions). In one embodiment, hypoxic cell cultureconditions include culturing cells at about less than 1% oxygen, aboutless than 2% oxygen, about less than 3% oxygen, about less than 4%oxygen, or about less than 5% oxygen. In another embodiment, normal ornormoxic culture conditions include culturing cells at about 10% oxygen,about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen,about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen,about 20% oxygen, or about 21% oxygen.

In one other embodiment, induction or increased expression of EPO isobtained and can be observed by culturing cells at about less than 5%available oxygen and comparing EPO expression levels to cells culturedat atmospheric (about 21%) oxygen. In another embodiment, the inductionof EPO is obtained in a culture of cells capable of expressing EPO by amethod that includes a first culture phase in which the culture of cellsis cultivated at atmospheric oxygen (about 21%) for some period of timeand a second culture phase in which the available oxygen levels arereduced and the same cells are cultured at about less than 5% availableoxygen. In another embodiment, the EPO expression that is responsive tohypoxic conditions is regulated by HIF1α. Those of ordinary skill in theart will appreciate that other oxygen manipulation culture conditionsknown in the art may be used for the cells described herein.

In one aspect, the formulation contains enriched populations ofEPO-producing mammalian cells characterized by bio-responsiveness (e.g.,EPO expression) to perfusion conditions. In one embodiment, theperfusion conditions include transient, intermittent, or continuousfluid flow (perfusion). In one embodiment, the EPO expression ismechanically-induced when the media in which the cells are cultured isintermittently or continuously circulated or agitated in such a mannerthat dynamic forces are transferred to the cells via the flow. In oneembodiment, the cells subjected to the transient, intermittent, orcontinuous fluid flow are cultured in such a manner that they arepresent as three-dimensional structures in or on a material thatprovides framework and/or space for such three-dimensional structures toform. In one embodiment, the cells are cultured on porous beads andsubjected to intermittent or continuous fluid flow by means of a rockingplatform, orbiting platform, or spinner flask. In another embodiment,the cells are cultured on three-dimensional scaffolding and placed intoa device whereby the scaffold is stationary and fluid flowsdirectionally through or across the scaffolding. Those of ordinary skillin the art will appreciate that other perfusion culture conditions knownin the art may be used for the cells described herein.

Cellular Aggregates

In one other aspect, the formulations of the present disclosure containcellular aggregates or spheroids. In one embodiment, the cellularaggregate comprises a bioactive cell population described herein. Inanother embodiment, the cellular aggregate comprises bioactive renalcells such as, for example, renal cell admixtures, enriched renal cellpopulations, and combinations of renal cell fractions.

In certain embodiments, the bioactive renal cells of the disclosure maybe cultured in 3D formats as described further herein. In someembodiments, the term “organoid” refers to an accumulation of cells,with a phenotype and/or function, consistent with a native kidney. Insome embodiments, organoids comprise mixed populations of cells, of avariety of lineages, which are typically found in vivo in a giventissue. In some embodiments, the organoids of this disclosure are formedin vitro, via any means, whereby the cells of the disclosure formaggregates, which in turn may form spheroids, organoids, or acombination thereof. Such aggregates, spheroids or organoids, in someembodiments, assume a structure consistent with a particular organ. Insome embodiments, such aggregates, spheroids or organoids, expresssurface markers, which are typically expressed by cells of theparticular organ. In some embodiments, such aggregates, spheroids ororganoids, produce compounds or materials, which are typically expressedby cells of the particular organ. In certain embodiments, the cells ofthe disclosure may be cultured on natural substrates, e.g., gelatin. Inother embodiments, the cells of the disclosure may be cultured onsynthetic substrates, e.g., PGLA.

Inactive Cell Populations

As described herein, certain subfractions of a heterogeneous populationof renal cells, enriched for bioactive components and depleted ofinactive or undesired components, provide superior therapeutic andregenerative outcomes than the starting population. In preferredembodiments, the formulations provided by the present disclosure containcellular populations that are depleted of B1 and/or B5 cell populations.For instance, the following may be depleted of B1 and/or 85: admixturesof two or more of B2, B3, and B4 (or B4′); an enriched cell populationof B2, B3, and B4 (or B4′).

The B1 cell population comprises large, granular cells of the collectingduct and tubular system, with the cells of the population having abuoyant density less than about 1.045 g/m. The B5 cell population iscomprised of debris and small cells of low granularity and viability andhaving a buoyant density greater than about 1.091 g/ml.

Methods of Isolating and Culturina Cell Populations

In one aspect, the formulations of the present disclosure contain cellpopulations that have been isolated and/or cultured from kidney tissue.Methods are provided herein for separating and isolating the renalcellular components, e.g., enriched cell populations that will be usedin the formulations for therapeutic use, including the treatment ofkidney disease, anemia, EPO deficiency, tubular transport deficiency,and glomerular filtration deficiency. In one embodiment, the cellpopulations are isolated from freshly digested, i.e., mechanically orenzymatically digested, kidney tissue or from heterogeneous in vitrocultures of mammalian kidney cells.

The formulations may contain heterogeneous mixtures of renal cells thathave been cultured in hypoxic culture conditions prior to separation ona density gradient provides for enhanced distribution and composition ofcells in both B4, including B4′, and B2 and/or B3 fractions. Theenrichment of oxygen-dependent cells in B4 from B2 was observed forrenal cells isolated from both diseased and non-diseased kidneys.Without wishing to be bound by theory, this may be due to one or more ofthe following phenomena: 1) selective survival, death, or proliferationof specific cellular components during the hypoxic culture period; 2)alterations in cell granularity and/or size in response to the hypoxicculture, thereby effecting alterations in buoyant density and subsequentlocalization during density gradient separation; and 3) alterations incell gene/protein expression in response to the hypoxic culture period,thereby resulting in differential characteristics of the cells withinany given fraction of the gradient. Thus, in one embodiment, theformulations contain cell populations enriched for tubular cells, e.g.,B2, are hypoxia-resistant.

Exemplary techniques for separating and isolating the cell populationsinclude separation on a density gradient based on the differentialspecific gravity of different cell types contained within the populationof interest. The specific gravity of any given cell type can beinfluenced by the degree of granularity within the cells, theintracellular volume of water, and other factors. In one aspect, thepresent disclosure provides optimal gradient conditions for isolation ofthe cell preparations, e.g., B2 and B4, including B4′, across multiplespecies including, but not limited to, human, canine, and rodent. In apreferred embodiment, a density gradient is used to obtain a novelenriched population of tubular cells fraction, i.e., B2 cell population,derived from a heterogeneous population of renal cells. In oneembodiment, a density gradient is used to obtain a novel enrichedpopulation of EPO-producing cells fraction, i.e., B4 cell population,derived from a heterogeneous population of renal cells. In otherembodiments, a density gradient is used to obtain enrichedsubpopulations of tubular cells, glomerular cells, and endothelial cellsof the kidney. In one embodiment, both the EPO-producing and the tubularcells are separated from the red blood cells and cellular debris. In oneembodiment, the EPO-producing, glomerular, and vascular cells areseparated from other cell types and from red blood cells and cellulardebris, while a subpopulation of tubular cells and collecting duct cellsare concomitantly separated from other cell types and from red bloodcells and cellular debris. In one other embodiment, the endocrine,glomerular, and/or vascular cells are separated from other cell typesand from red blood cells and cellular debris, while a subpopulation oftubular cells and collecting duct cells are concomitantly separated fromother cell types and from red blood cells and cellular debris.

In one aspect, the formulations of the present disclosure contain cellpopulations generated by using, in part, the OPTIPREP® (Axis-Shield)density gradient medium, comprising 60% nonionic iodinated compoundiodixanol in water, based on certain key features described below. Oneof skill in the art, however, will recognize that any density gradientor other means, e.g., immunological separation using cell surfacemarkers known in the art, comprising necessary features for isolatingthe cell populations of the instant disclosure may be used. It shouldalso be recognized by one skilled in the art that the same cellularfeatures that contribute to separation of cellular subpopulations viadensity gradients (size and granularity) can be exploited to separatecellular subpopulations via flow cytometry (forward scatter=a reflectionof size via flow cytometry, and side scatter=a reflection ofgranularity). Importantly, the density gradient medium should have lowtoxicity towards the specific cells of interest. While the densitygradient medium should have low toxicity toward the specific cells ofinterest, the instant disclosure contemplates the use of gradientmediums which play a role in the selection process of the cells ofinterest. Without wishing to be bound by theory, it appears that thecell populations disclosed herein recovered by the gradient comprisingiodixanol are iodixanol-resistant, as there is an appreciable loss ofcells between the loading and recovery steps, suggesting that exposureto iodixanol under the conditions of the gradient leads to eliminationof certain cells. The cells appearing in the specific bands after theiodixanol gradient are resistant to any untoward effects of iodixanoland/or density gradient exposure. Accordingly, the use of additionalcontrast media which are mild to moderate nephrotoxins in the isolationand/or selection of the cell populations for the formulations describedherein is also contemplated. In addition, the density gradient mediumshould also not bind to proteins in human plasma or adversely affect keyfunctions of the cells of interest.

In another aspect, the present disclosure provides formulationscontaining cell populations that have been enriched and/or depleted ofkidney cell types using fluorescent activated cell sorting (FACS). Inone embodiment, kidney cell types may be enriched and/or depleted usingBD FACSAria™ or equivalent.

In another aspect, the formulations contain cell populations that havebeen enriched and/or depleted of kidney cell types using magnetic cellsorting. In one embodiment, kidney cell types may be enriched and/ordepleted using the Miltenyi autoMACS® system or equivalent.

In another aspect, the formulations may include renal cell populationsthat have been subject to three-dimensional culturing. In one aspect,the methods of culturing the cell populations are via continuousperfusion. In one embodiment, the cell populations cultured viathree-dimensional culturing and continuous perfusion demonstrate greatercellularity and interconnectivity when compared to cell populationscultured statically. In another embodiment, the cell populationscultured via three dimensional culturing and continuous perfusiondemonstrate greater expression of EPO, as well as enhanced expression ofrenal tubule-associate genes such as e-cadherin when compared to staticcultures of such cell populations. In yet another embodiment, the cellpopulations cultured via continuous perfusion demonstrate greater levelsof glucose and glutamine consumption when compared to cell populationscultured statically.

As described herein, low or hypoxic oxygen conditions may be used in themethods to prepare the cell populations for the formulations providedfor herein. However, the methods of preparing cell populations may beused without the step of low oxygen conditioning. In one embodiment,normoxic conditions may be used.

Those of ordinary skill in the art will appreciate that other methods ofisolation and culturing known in the art may be used for the cellsdescribed herein.

3. Biomaterials

A variety of biomaterials may be combined with an active agent toprovide the therapeutic formulations of the present disclosure. Thebiomaterials may be in any suitable shape (e.g., beads) or form (e.g.,liquid, gel, etc.). As described in Bertram et al. U.S. PublishedApplication 20070276507 (incorporated herein by reference in itsentirety), polymeric matrices or scaffolds may be shaped into any numberof desirable configurations to satisfy any number of overall system,geometry or space restrictions. In one embodiment, the matrices orscaffolds of the present disclosure may be three-dimensional and shapedto conform to the dimensions and shapes of an organ or tissue structure.For example, in the use of the polymeric scaffold for treating kidneydisease, anemia, EPO deficiency, tubular transport deficiency, orglomerular filtration deficiency, a three-dimensional (3-D) matrix maybe used. A variety of differently shaped 3-D scaffolds may be used.Naturally, the polymeric matrix may be shaped in different sizes andshapes to conform to differently sized patients. The polymeric matrixmay also be shaped in other ways to accommodate the special needs of thepatient. In another embodiment, the polymeric matrix or scaffold may bea biocompatible, porous polymeric scaffold. The scaffolds may be formedfrom a variety of synthetic or naturally-occurring materials including,but not limited to, open-cell polylactic acid (OPLA®), cellulose ether,cellulose, cellulosic ester, fluorinated polyethylene, phenolic,poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,polyester, polyestercarbonate, polyether, polyetheretherketone,polyetherimide, polyetherketone, polyethersulfone, polyethylene,polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenyleneoxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, collagens, gelatin, alginate, laminins,fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, orcopolymers or physical blends thereof. Scaffolding configurations mayrange from liquid suspensions to soft porous scaffolds to rigid,shape-holding porous scaffolds. In one embodiment, the configuration isthe liquid form of a solution that is capable of becoming a hydrogel.

Hydrogels may be formed from a variety of polymeric materials and areuseful in a variety of biomedical applications. Hydrogels can bedescribed physically as three-dimensional networks of hydrophilicpolymers. Depending on the type of hydrogel, they contain varyingpercentages of water, but altogether do not dissolve in water. Despitetheir high water content, hydrogels are capable of additionally bindinggreat volumes of liquid due to the presence of hydrophilic residues.Hydrogels swell extensively without changing their gelatinous structure.The basic physical features of hydrogel can be specifically modified,according to the properties of the polymers used and the additionalspecial equipments of the products.

Preferably, the hydrogel is made of a polymer, a biologically derivedmaterial, a synthetically derived material or combinations thereof, thatis biologically inert and physiologically compatible with mammaliantissues. The hydrogel material preferably does not induce aninflammatory response. Examples of other materials which can be used toform a hydrogel include (a) modified alginates, (b) polysaccharides(e.g. gellan gum and carrageenans) which gel by exposure to monovalentcations, (c) polysaccharides (e.g., hyaluronic acid) that are veryviscous liquids or are thixotropic and form a gel over time by the slowevolution of structure, (d) gelatin or collagen, and (e) polymerichydrogel precursors (e.g., polyethylene oxide-polypropylene glycol blockcopolymers and proteins). U.S. Pat. No. 6,224,893 B1 provides a detaileddescription of the various polymers, and the chemical properties of suchpolymers, that are suitable for making hydrogels.

Scaffolding or biomaterial characteristics may enable cells to attachand interact with the scaffolding or biomaterial material, and/or mayprovide porous spaces into which cells can be entrapped. In oneembodiment, the porous scaffolds or biomaterials allow for the additionor deposition of one or more populations or admixtures of cells on abiomaterial configured as a porous scaffold (e.g., by attachment of thecells) and/or within the pores of the scaffold (e.g., by entrapment ofthe cells). In another embodiment, the scaffolds or biomaterials allowor promote for cell:cell and/or cell:biomaterial interactions within thescaffold to form constructs as described herein.

In one embodiment, the biomaterial is comprised of hyaluronic acid (HA)in hydrogel form, containing HA molecules ranging in size from 5.1 kDAto >2×10⁶ kDa. In another embodiment, the biomaterial is comprised ofhyaluronic acid in porous foam form, also containing HA moleculesranging in size from 5.1 kDA to >2×10⁶ kDa. In yet another embodiment,the biomaterial is comprised of a poly-lactic acid (PLA)-based foam,having an open-cell structure and pore size of about 50 microns to about300 microns. In yet another embodiment, the specific cell populations,preferentially B2 but also B4, provide directly and/or stimulatesynthesis of high molecular weight Hyaluronic Acid through HyaluronicAcid Synthase-2 (HAS-2), especially after intra-renal implantation.

The biomaterials described herein may also be designed or adapted torespond to certain external conditions, e.g., in vitro or in vivo. Inone embodiment, the biomaterials are temperature-sensitive (e.g., eitherin vitro or in vivo). In another embodiment, the biomaterials areadapted to respond to exposure to enzymatic degradation (e.g., either invitro or in vivo). The biomaterials' response to external conditions canbe fine tuned as described herein. Temperature sensitivity of theformulation described can be varied by adjusting the percentage of abiomaterial in the formulation. For example, the percentage of gelatinin a solution can be adjusted to modulate the temperature sensitivity ofthe gelatin in the final formulation (e.g., liquid, gel, beads, etc.).Alternatively, biomaterials may be chemically cross-linked to providegreater resistance to enzymatic degradation. For instance, acarbodiimide crosslinker may be used to chemically crosslink gelatinbeads thereby providing a reduced susceptibility to endogenous enzymes.

In one aspect, the response by the biomaterial to external conditionsconcerns the loss of structural integrity of the biomaterial. Althoughtemperature-sensitivity and resistance to enzymatic degradation areprovided above, other mechanisms exist by which the loss of materialintegrity may occur in different biomaterials. These mechanisms mayinclude, but are not limited to thermodynamic (e.g., a phase transitionsuch as melting, diffusion (e.g., diffusion of an ionic crosslinker froma biomaterial into the surrounding tissue)), chemical, enzymatic, pH(e.g., pH-sensitive liposomes), ultrasound, and photolabile (lightpenetration). The exact mechanism by which the biomaterial losesstructural integrity will vary but typically the mechanism is triggeredeither at the time of implantation or post-implantation.

Those of ordinary skill in the art will appreciate that other types ofsynthetic or naturally-occurring materials known in the art may be usedto form scaffolds as described herein.

In one aspect, the constructs as described herein are made from theabove-referenced scaffolds or biomaterials.

4. Constructs

In one aspect, the disclosure provides formulations that containimplantable constructs having one or more of the cell populationsdescribed herein for the treatment of kidney disease, anemia, or EPOdeficiency in a subject in need. In one embodiment, the construct ismade up of a biocompatible material or biomaterial, scaffold or matrixcomposed of one or more synthetic or naturally-occurring biocompatiblematerials and one or more cell populations or admixtures of cellsdescribed herein deposited on or embedded in a surface of the scaffoldby attachment and/or entrapment. In certain embodiments, the constructis made up of a biomaterial and one or more cell populations oradmixtures of cells described herein coated with, deposited on,deposited in, attached to, entrapped in, embedded in, seeded, orcombined with the biomaterial component(s). Any of the cell populationsdescribed herein, including enriched cell populations or admixturesthereof, may be used in combination with a matrix to form a construct.

In one aspect, the formulation contains constructs that are made up ofbiomaterials designed or adapted to respond to external conditions asdescribed herein. As a result, the nature of the association of the cellpopulation with the biomaterial in a construct will change dependingupon the external conditions. For example, a cell population'sassociation with a temperature-sensitive biomaterial varies withtemperature. In one embodiment, the construct contains a cell populationand biomaterial having a substantially solid state at about 8° C. orlower and a substantially liquid state at about ambient temperature orabove, wherein the cell population is suspended in the biomaterial atabout 8° C. or lower.

However, the cell population is substantially free to move throughoutthe volume of the biomaterial at about ambient temperature or above.Having the cell population suspended in the substantially solid phase ata lower temperature provides stability advantages for the cells, such asfor anchorage-dependent cells, as compared to cells in a fluid.Moreover, having cells suspended in the substantially solid stateprovides one or more of the following benefits: i) prevents settling ofthe cells, ii) allows the cells to remain anchored to the biomaterial ina suspended state; iii) allows the cells to remain more uniformlydispersed throughout the volume of the biomaterial; iv) prevents theformation of cell aggregates; and v) provides better protection for thecells during storage and transportation of the formulation. Aformulation that can retain such features leading up to theadministration to a subject is advantageous at least because the overallhealth of the cells in the formulation will be better and a more uniformand consistent dosage of cells will be administered.

In another embodiment, the deposited cell population or cellularcomponent of the construct is a first kidney cell population enrichedfor oxygen-tunable EPO-producing cells. In another embodiment, the firstkidney cell population contains glomerular and vascular cells inaddition to the oxygen-tunable EPO-producing cells. In one embodiment,the first kidney cell population is a B4′ cell population. In one otherembodiment, the deposited cell population or cellular component(s) ofthe construct includes both the first enriched renal cell population anda second renal cell population. In some embodiments, the second cellpopulation is not enriched for oxygen-tunable EPO producing cells. Inanother embodiment, the second cell population is enriched for renaltubular cells. In another embodiment, the second cell population isenriched for renal tubular cells and contains collecting duct epithelialcells. In other embodiments, the renal tubular cells are characterizedby the expression of one or more tubular cell markers that may include,without limitation, megalin, cubilin, hyaluronic acid synthase 2 (HAS2),Vitamin D3 25-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin(Ecad), Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RASoncogene family (Rab17), GATA binding protein 3 (Gata3), FXYDdomain-containing ion transport regulator 4 (Fxyd4), solute carrierfamily 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehydedehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1family, member A3 (Aldh1a3), and Calpain-8 (Capn8).

In one embodiment, the cell populations deposited on or combined withbiomaterials or scaffolds to form constructs are derived from a varietyof sources, such as autologous sources. Non-autologous sources are alsosuitable for use, including without limitation, allogeneic, or syngeneic(autogeneic or isogeneic) sources.

Those of ordinary skill in the art will appreciate there are severalsuitable methods for depositing or otherwise combining cell populationswith biomaterials to form a construct.

In one aspect, the constructs are suitable for use in the methods of usedescribed herein. In one embodiment, the constructs are suitable foradministration to a subject in need of treatment for a kidney disease ofany etiology, anemia, or EPO deficiency of any etiology. In otherembodiments, the constructs are suitable for administration to a subjectin need of an improvement in or restoration of erythroid homeostasis. Inanother embodiment, the constructs are suitable for administration to asubject in need of improved kidney function.

In yet another aspect, the present disclosure provides a construct forimplantation into a subject in need of improved kidney functioncomprising: a) a biomaterial comprising one or more biocompatiblesynthetic polymers or naturally-occurring proteins or peptides; and b)an admixture of mammalian renal cells derived from a subject havingkidney disease comprising a first cell population, B2, comprising anisolated, enriched population of tubular cells having a density between1.045 g/mL and 1.052 g/mL and a second cell population, B4′, comprisingerythropoietin (EPO)-producing cells and vascular cells but depleted ofglomerular cells having a density between 1.063 g/mL and 1.091 g/mL,coated with, deposited on or in, entrapped in, suspended in, embedded inand/or otherwise combined with the biomaterial. In certain embodiments,the admixture does not include a B1 cell population comprising largegranular cells of the collecting duct and tubular system having adensity of <1.045 g/ml, or a B5 cell population comprising debris andsmall cells of low granularity and viability with a density >1.091 g/ml.

In one embodiment, the construct includes a B4′ cell population which ischaracterized by expression of a vascular marker. In some embodiments,the B4′ cell population is not characterized by expression of aglomerular marker. In certain embodiments, the admixture is capable ofoxygen-tunable erythropoietin (EPO) expression. In all embodiments, theadmixture may be derived from mammalian kidney tissue or cultured kidneycells.

In one embodiment, the construct includes a biomaterial configured as athree-dimensional (3-D) porous biomaterial suitable for entrapmentand/or attachment of the admixture. In another embodiment, the constructincludes a biomaterial configured as a liquid or semi-liquid gelsuitable for embedding, attaching, suspending, or coating mammaliancells. In yet another embodiment, the construct includes a biomaterialconfigured comprised of a predominantly high-molecular weight species ofhyaluronic acid (HA) in hydrogel form. In another embodiment, theconstruct includes a biomaterial comprised of a predominantlyhigh-molecular weight species of hyaluronic acid in porous foam form. Inyet another embodiment, the construct includes a biomaterial comprisedof a poly-lactic acid-based foam having pores of between about 50microns to about 300 microns. In still another embodiment, the constructincludes one or more cell populations that may be derived from a kidneysample that is autologous to the subject in need of improved kidneyfunction. In certain embodiments, the sample is a kidney biopsy. In someembodiments, the subject has a kidney disease. In yet other embodiments,the cell population is derived from a non-autologous kidney sample. Inone embodiment, the construct provides erythroid homeostasis.

5. Phenotypic Characterization of Renal Cells

The cells isolated at any stage of the process may be characterized bytheir phenotype. In one embodiment, the cells are a heterogeneous renalcell population that has been enriched. In a further embodiment, theenriched heterogeneous renal cell population has been cultured underhypoxic conditions for at least 24 hours. In a yet further embodiment,the enriched heterogeneous renal cell population has been subjected to adensity gradient.

The presence (e.g., expression) and/or level/amount of variousbiomarkers in a sample can be analyzed by a number of methodologies,many of which are known in the art and understood by the skilledartisan, including, but not limited to, immunohistochemical (“IHC”),Western blot analysis, immunoprecipitation, molecular binding assays,ELISA, ELIFA, fluorescence activated cell sorting (“FACS”), MassARRAY,proteomics, biochemical enzymatic activity assays, in situhybridization, Southern analysis, Northern analysis, whole genomesequencing, polymerase chain reaction (“PCR”) including quantitativereal time PCR (“qRT-PCR”) and other amplification type detectionmethods, such as, for example, branched DNA, SISBA, TMA and the like),RNA-Seq, FISH, microarray analysis, gene expression profiling, and/orserial analysis of gene expression (“SAGE”), as well as any one of thewide variety of assays that can be performed by protein, gene, and/ortissue array analysis. Typical protocols for evaluating the status ofgenes and gene products are found, for example in Ausubel et al., eds.,1995, Current Protocols In Molecular Biology, Units 2 (NorthernBlotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCRAnalysis). Multiplexed immunoassays such as those available from RulesBased Medicine or Meso Scale Discovery may also be used.

In one aspect, a method of detecting the presence of two or morebiomarkers in a heterogeneous renal cell sample is provided, the methodcomprising contacting the sample with an antibody directed to abiomarker under conditions permissive for binding of the antibody to itscognate ligand (i.e., biomarker), and detecting the presence of thebound antibody, e.g., by detecting whether a complex is formed betweenthe antibody and the biomarker. In some embodiments, the detection ofthe presence of one or more biomarkers is by immunohistochemistry.

In certain embodiments, any of the antibodies provided herein is usefulfor detecting the presence of a biomarker in a heterogeneous renal cellsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a SRC sample.

In certain embodiments, the heterogeneous renal cells are identifiedwith one or more reagents that allow detection of a biomarker selectedfrom AQP1, AQP2, AQP4, Calbindin, Calponin, CD117, CD133, CD146, CD24,CD31 (PECAM-1), CD54 (ICAM-1), CD73, CK18, CK19, CK40 to 67, CK7, CK8,CK8/18, CK8/18/19, Connexin 43, Cubilin, CXCR4 (Fusin), DBA, E-cadherin(CD324), EPO (erythropoeitin), GGT1, GLEPP1 (glomerular epithelialprotein 1), Haptoglobulin, Itgb1 (Integrin β1), KIM-1/TIM-1 (kidneyinjury molecule-1/T-cell immunoglobulin and mucin-containing molecule),MAP-2 (microtubule-associated protein 2), Megalin, N-cadherin, Nephrin,NKCC (Na—K—Cl-cotransporters), OAT-1 (organic anion transporter 1),Osteopontin, Pan-cadherin, PCLP1 (podocalyxin-like 1 molecule), Podocin,SMA (smooth muscle alpha-actin), Synaptopodin, THP (tamm-horsfallprotein), Vimentin, and αGST-1 (alpha glutathione 5-transferase). Incertain embodiments, a biomarker is detected by monoclonal or polyclonalantibodies.

In one embodiment, an detectable label comprises a radioactive atom toform a radioconjugate. A variety of radioactive isotopes are availablefor the production of radioconjugates. Examples include ²¹¹At, ¹³¹I,¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ²¹²Pb and radioactiveisotopes of Lu. When the radioconjugate is used for detection, it maycomprise a radioactive atom for scintigraphic studies, for example⁹⁹Tc-m (metastable nuclear isomer) or ¹²³I, or a spin label for nuclearmagnetic resonance (NMR) imaging (also known as magnetic resonanceimaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

In case more than one detectable label (including a dye) is used in onetesting, it is preferred that the detectable labels are selected suchthat each label can be independently detected without substantialinterference to any other detectable signals present in the sample. Forexample, the detectable labels (including a dye) may be differentfluorescent molecules showing different colors under the detectioncondition.

The detection can be carried out by any suitable method, for example,those based on immunofluorescent microscopy, flow cytometry, fiber-opticscanning cytometry, or laser scanning cytometry.

In some embodiments, the expression of a biomarker in a cell isdetermined by evaluating mRNA in a cell. Methods for the evaluation ofmRNAs in cells are well known and include, for example, hybridizationassays using complementary DNA probes (such as in situ hybridizationusing labeled riboprobes specific for the one or more genes, Northernblot and related techniques) and various nucleic acid amplificationassays (such as RT-PCR using complementary primers specific for one ormore of the genes, and other amplification type detection methods, suchas, for example, branched DNA, SISBA, TMA and the like). In someembodiments, the expression of a biomarker in a test sample is comparedto a reference sample. For example, the test sample may be a diseasedtissue sample and the reference sample may be from normal tissue.

Samples from mammals can be conveniently assayed for mRNAs usingNorthern, dot blot or PCR analysis. In addition, such methods caninclude one or more steps that allow one to determine the levels oftarget mRNA in a biological sample (e.g., by simultaneously examiningthe levels a comparative control mRNA sequence of a “housekeeping” genesuch as an actin family member). Optionally, the sequence of theamplified target cDNA can be determined.

Optional methods include protocols which examine or detect mRNAs, suchas target mRNAs, in a tissue or cell sample by microarray technologies.Using nucleic acid microarrays, test and control mRNA samples from testand control samples are reverse transcribed and labeled to generate cDNAprobes. The probes are then hybridized to an array of nucleic acidsimmobilized on a solid support. The array is configured such that thesequence and position of each member of the array is known. For example,a selection of genes whose expression correlates with a cell populationcapable of eliciting a regenerative response may be arrayed on a solidsupport. Hybridization of a labeled probe with a particular array memberindicates that the sample from which the probe was derived expressesthat gene.

According to some embodiments, presence and/or level/amount is measuredby observing protein expression levels of an aforementioned gene. Incertain embodiments, the method comprises contacting the biologicalsample with antibodies to a biomarker described herein under conditionspermissive for binding of the biomarker, and detecting whether a complexis formed between the antibodies and biomarker.

In certain embodiments, the presence and/or level/amount of biomarkerproteins in a sample are examined using IHC and staining protocols. IHCstaining of cells has been shown to be a reliable method of determiningor detecting the presence of proteins in a sample. In one aspect, levelof biomarker is determined using a method comprising: (a) performing IHCanalysis of a sample (such as a renal cell sample) with an antibody; andb) determining level of a biomarker in the sample. In some embodiments,IHC staining intensity is determined relative to a reference value.

IHC may be performed in combination with additional techniques such asmorphological staining and/or fluorescence in-situ hybridization. Twogeneral methods of IHC are available; direct and indirect assays.According to the first assay, binding of antibody to the target antigenis determined directly. This direct assay uses a labeled reagent, suchas a fluorescent tag or an enzyme-labeled primary antibody, which can bevisualized without further antibody interaction. In a typical indirectassay, unconjugated primary antibody binds to the antigen and then alabeled secondary antibody binds to the primary antibody. Where thesecondary antibody is conjugated to an enzymatic label, a chromogenic orfluorogenic substrate is added to provide visualization of the antigen.Signal amplification occurs because several secondary antibodies mayreact with different epitopes on the primary antibody.

The primary and/or secondary antibody used for IHC typically will belabeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories: (a)Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I; (b) colloidal goldparticles; (c) fluorescent labels including, but are not limited to,rare earth chelates (europium chelates), Texas Red, rhodamine,fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin,phycocyanin, or commercially available fluorophores such SPECTRUMORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of theabove; (d) various enzyme-substrate labels are available and U.S. Pat.No. 4,275,149 provides a review of some of these. Examples of enzymaticlabels include luciferases (e.g., firefly luciferase and bacterialluciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRP), alkaline phosphatase,1-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like.

Examples of enzyme-substrate combinations include, for example,horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate;alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenicsubstrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate(e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g.,4-methylumbelliferyl-β-D-galactosidase). For a general review of these,see U.S. Pat. Nos. 4,275,149 and 4,318,980.

In an exemplary method, the sample may be contacted with an antibodyspecific for the biomarker under conditions sufficient for anantibody-biomarker complex to form, and then detecting the complex. Thepresence of the biomarker may be detected in a number of ways, such asby Western blotting and ELISA procedures for assaying a wide variety oftissues and samples, including plasma or serum. A wide range ofimmunoassay techniques using such an assay format are available, see,e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These includeboth single-site and two-site or “sandwich” assays of thenon-competitive types, as well as in the traditional competitive bindingassays. These assays also include direct binding of a labeled antibodyto a target biomarker.

The presence and/or level/amount of a selected biomarker in a tissue orcell sample may also be examined by way of functional or activity-basedassays. For instance, if the biomarker is an enzyme, one may conductassays known in the art to determine or detect the presence of the givenenzymatic activity in the tissue or cell sample.

In certain embodiments, the samples are normalized for both differencesin the amount of the biomarker assayed and variability in the quality ofthe samples used, and variability between assay runs. Such normalizationmay be accomplished by detecting and incorporating the level of certainnormalizing biomarkers, including well known housekeeping genes, such asACTB. Alternatively, normalization can be based on the mean or mediansignal of all of the assayed genes or a large subset thereof (globalnormalization approach). On a gene-by-gene basis, measured normalizedamount of a subject tumor mRNA or protein is compared to the amountfound in a reference set. Normalized expression levels for each mRNA orprotein per tested tumor per subject can be expressed as a percentage ofthe expression level measured in the reference set. The presence and/orexpression level/amount measured in a particular subject sample to beanalyzed will fall at some percentile within this range, which can bedetermined by methods well known in the art.

In embodiments, the cytokeratin is selected from CK8, CK18, CK19 andcombinations thereof. In certain embodiments, the cytokeratin is CK8,CK18, CK19, CK8/CK18, CK8/CK19, CK18/CK19 or CK8/CK18/CK19, wherein the“/” refers to a combination of the cytokeratins adjacent thereto. In allembodiments, the cytokeratin has a level of expression greater thanabout 80%, about 85%, about 90%, or about 95%.

In embodiments, the GGT is GGT-1. In all embodiments the GGT has a levelof expression greater than about 10%, about 15%, about 18%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, or about 60%.

6. Methods of Use

In another aspect, the formulations of the present disclosure may beadministered for the treatment of disease. For example, bioactive cellsmay be administered to a native organ as part of a formulation describedherein. In one embodiment, the bioactive cells may be sourced from thenative organ that is the subject of the administration or from a sourcethat is not the target native organ.

In one aspect, the present disclosure provides methods for the treatmentof a kidney disease, anemia, or EPO deficiency in a subject in need withthe formulations containing kidney cell populations and admixtures ofkidney cells as described herein. In one embodiment, the methodcomprises administering to the subject a formulation containing acomposition that includes a first kidney cell population enriched forEPO-producing cells. In another embodiment, the first cell population isenriched for EPO-producing cells, glomerular cells, and vascular cells.In one embodiment, the first kidney cell population is a B4′ cellpopulation. In another embodiment, the composition may further includeone or more additional kidney cell populations. In one embodiment, theadditional cell population is a second cell population not enriched forEPO-producing cells. In another embodiment, the additional cellpopulation is a second cell population not enriched for EPO-producingcells, glomerular cells, or vascular cells. In another embodiment, thecomposition also includes a kidney cell population or admixture ofkidney cells deposited in, deposited on, embedded in, coated with,suspended in, or entrapped in a biomaterial to form an implantableconstruct, as described herein, for the treatment of a disease ordisorder described herein. In one embodiment, the cell populations areused alone or in combination with other cells or biomaterials, e.g.,hydrogels, porous scaffolds, or native or synthetic peptides orproteins, to stimulate regeneration in acute or chronic disease states.

In another aspect, the effective treatment of a kidney disease in asubject by the methods disclosed herein can be observed through variousindicators of erythropoiesis and/or kidney function. In one embodiment,the indicators of erythroid homeostasis include, without limitation,hematocrit (HCT), hemoglobin (HB), mean corpuscular hemoglobin (MCH),red blood cell count (RBC), reticulocyte number, reticulocyte %, meancorpuscular volume (MCV), and red blood cell distribution width (RDW).In one other embodiment, the indicators of kidney function include,without limitation, serum albumin, albumin to globulin ratio (A/Gratio), serum phosphorous, serum sodium, kidney size (measurable byultrasound), serum calcium, phosphorous:calcium ratio, serum potassium,proteinuria, urine creatinine, serum creatinine, blood nitrogen urea(BUN), cholesterol levels, triglyceride levels and glomerular filtrationrate (GFR). Furthermore, several indicators of general health andwell-being include, without limitation, weight gain or loss, survival,blood pressure (mean systemic blood pressure, diastolic blood pressure,or systolic blood pressure), and physical endurance performance.

In another embodiment, an effective treatment with a bioactive renalcell formulation is evidenced by stabilization of one or more indicatorsof kidney function. The stabilization of kidney function is demonstratedby the observation of a change in an indicator in a subject treated by amethod provided for herein as compared to the same indicator in asubject that has not been treated by the method herein. Alternatively,the stabilization of kidney function may be demonstrated by theobservation of a change in an indicator in a subject treated by a methodherein as compared to the same indicator in the same subject prior totreatment. The change in the first indicator may be an increase or adecrease in value. In one embodiment, the treatment provided by thepresent disclosure may include stabilization of blood urea nitrogen(BUN) levels in a subject where the BUN levels observed in the subjectare lower as compared to a subject with a similar disease state who hasnot been treated by the methods of the present disclosure. In one otherembodiment, the treatment may include stabilization of serum creatininelevels in a subject where the serum creatinine levels observed in thesubject are lower as compared to a subject with a similar disease statewho has not been treated by the methods of the present disclosure. Inanother embodiment, the treatment may include stabilization ofhematocrit (HCT) levels in a subject where the HCT levels observed inthe subject are higher as compared to a subject with a similar diseasestate who has not been treated by the methods of the present disclosure.In another embodiment, the treatment may include stabilization of redblood cell (RBC) levels in a subject where the RBC levels observed inthe subject are higher as compared to a subject with a similar diseasestate who has not been treated by the methods of the present disclosure.Those of ordinary skill in the art will appreciate that one or moreadditional indicators described herein or known in the art may bemeasured to determine the effective treatment of a kidney disease in thesubject.

In another aspect, the present disclosure concerns formulations for usein methods of providing erythroid homeostasis in a subject. In oneembodiment, the method includes the step of (a) administering to thesubject a formulation containing a renal cell population, e.g., B2 orB4′, or admixture of renal cells, e.g., B2/B4′ and/or B2/B3, or anenriched renal cell population, as described herein; and (b)determining, in a biological sample from the subject, that the level ofan erythropoiesis indicator is different relative to the indicator levelin a control, wherein the difference in indicator level (i) indicatesthe subject is responsive to the administering step (a), or (ii) isindicative of erythroid homeostasis in the subject. In anotherembodiment, the method includes the step of (a) administering to thesubject a formulation comprising a renal cell population or admixture ofrenal cells as described herein; and (b) determining, in a biologicalsample from the subject, that the level of an erythropoiesis indicatoris different relative to the indicator level in a control, wherein thedifference in indicator level (i) indicates the subject is responsive tothe administering step (s), or (ii) is indicative of erythroidhomeostasis in the subject. In another embodiment, the method includesthe step of (a) providing a biomaterial or biocompatible polymericscaffold; (b) depositing a renal cell population or admixture of renalcells of the present disclosure on or within the biomaterial or scaffoldin a manner described herein to form an implantable construct; (c)preparing a formulation containing the construct; (d) implanting theconstruct into the subject; and (e) determining, in a biological samplefrom the subject, that the level of an erythropoiesis indicator isdifferent relative to the indicator level in a control, wherein thedifference in indicator level (i) indicates the subject is responsive tothe administering step (a), or (ii) is indicative of erythroidhomeostasis in the subject.

In another aspect, the present disclosure concerns formulations for usein methods of providing both stabilization of kidney function andrestoration of erythroid homeostasis to a subject in need, said subjecthaving both a deficit in kidney function and an anemia and/orEPO-deficiency. In one embodiment, the method includes the step ofadministering a formulation containing a renal cell population oradmixture of renal cells as described herein that contain at least oneof the following cell types: tubular-derived cells, glomerulus-derivedcells, insterstitium-derived cells, collecting duct-derived cells,stromal tissue-derived cells, or cells derived from the vasculature. Inanother embodiment, the population or admixture contains bothEPO-producing cells and tubular epithelial cells, the tubular cellshaving been identified by at least one of the following markers:megalin, cubilin, hyaluronic acid synthase 2 (HAS2), Vitamin D325-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad),Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogenefamily (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containingion transport regulator 4 (Fxyd4), solute carrier family 9(sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3(Aldh1a3), and Calpain-8 (Capn8). In this embodiment, treatment of thesubject would be demonstrated by an improvement in at least oneindicator of kidney function concomitant with improvement in at leastone indicator of erythropoiesis, compared to either an untreated subjector to the subject's pre-treatment indicators.

In one aspect, the present disclosure provides formulations for use inmethods of (i) treating a kidney disease, anemia, or an EPO-deficiency;(ii) stabilizing kidney function, (iii) restoring erythroid homeostasis,or (iv) any combination of thereof by administering a renal cellpopulation enriched for EPO-producing cells or admixture of renal cellscontaining a cell population enriched for EPO-producing cells asdescribed herein, wherein the beneficial effects of the administrationare greater than the effects of administering a cell population notenriched for EPO-producing cells. In another embodiment, the enrichedcell population provides an improved level of serum blood urea nitrogen(BUN). In another embodiment, the enriched cell population provides animproved retention of protein in the serum. In another embodiment, theenriched cell population provides improved levels of serum cholesteroland/or triglycerides. In another embodiment, the enriched cellpopulation provides an improved level of Vitamin D. In one embodiment,the enriched cell population provides an improved phosphorus:calciumratio as compared to a non-enriched cell population. In anotherembodiment, the enriched cell population provides an improved level ofhemoglobin as compared to a non-enriched cell population. In a furtherembodiment, the enriched cell population provides an improved level ofserum creatinine as compared to a non-enriched cell population. In yetanother embodiment, the enriched cell population provides an improvedlevel of hematocrit as compared to a non-enriched cell population. In afurther embodiment, the enriched cell population provides an improvedlevel of red blood cell number (RBC#) as compared to a non-enriched cellpopulation. In one embodiment, the improved level of hematocrit isrestored to 95% normal healthy level. In a further embodiment, theenriched cell population provides an improved reticulocyte number ascompared to a non-enriched cell population. In other embodiments, theenriched cell population provides an improved reticulocyte percentage ascompared to a non-enriched cell population. In yet other embodiments,the enriched cell population provides an improved level of red bloodcell volume distribution width (RDW) as compared to a non-enriched cellpopulation. In yet another embodiment, the enriched cell populationprovides an improved level of hemoglobin as compared to a non-enrichedcell population. In yet another embodiment, the enriched cell populationprovides an erythroietic response in the bone marrow, such that themarrow cellularity is near-normal and the myeloid:erythroid ratio isnear normal.

In another aspect, the present disclosure provides formulations for usein methods of (i) treating a kidney disease, anemia, or anEPO-deficiency; (ii) stabilizing kidney function, (iii) restoringerythroid homeostasis, or (iv) any combination of thereof byadministering an enriched cell population, wherein the beneficialeffects of administering a renal cell population or admixture of renalcell populations described herein are characterized by improvederythroid homeostasis when compared to the beneficial effects providedby the administering of recombinant EPO (rEPO). In one embodiment, thepopulation or admixture, when administered to a subject in need providesimproved erythroid homeostasis (as determined by hematocrit, hemoglobin,or RBC#) when compared to the administration of recombinant EPO protein.In one embodiment, the population or admixture, when administeredprovides an improved level of hematocrit, RBC, or hemoglobin as comparedto recombinant EPO, being no greater than about 10% lower or higher thanhematocrit in a control. In a further embodiment, a single dose ordelivery of the population or admixture, when administered providesimprovement in erythroid homeostasis (as determined by increase inhematocrit, hemoglobin, or RBC#) in the treated subject for a period oftime that significantly exceeds the period of time that a single dose ordelivery of the recombinant EPO protein provides improvement inerythroid homeostasis. In another embodiment, the population oradmixture, when administered at a dose described herein does not resultin hematocrit, hemoglobin, or RBC#greater than about 110% of normallevels in matched healthy controls. In a further embodiment, thepopulation or admixture, when administered at a dose described hereinprovides superior erythroid homeostasis (as determined by hematocrit,hemoglobin, or RBC#) compared to recombinant EPO protein delivered at adose described herein. In another embodiment, the recombinant EPO isdelivered at a dose of about 100 IU/kg, about 200 IU/kg, about 300IU/kg, about 400 IU/kg, or about 500 IU/kg. Those of ordinary skill inthe art will appreciate that other dosages of recombinant EPO known inthe art may be suitable.

Another embodiment of the present disclosure is directed to the use offormulations containing at least one cell population, including enrichedcell populations and admixtures thereof, described herein, or animplantable construct described herein, or secreted products asdescribed herein, for the preparation of a medicament for the treatmentof a kidney disease, anemia, or EPO deficiency in a subject in need, theproviding of erythroid homeostasis in a subject in need, the improvementof kidney function in a subject in need, or providing a regenerativeeffect to a native kidney.

Another embodiment of the present disclosure is directed to formulationscontaining specific enriched cell population(s) (described herein) forthe treatment of a kidney disease of a specific etiology, based onselection of specific cell subpopulation(s) based on specific verifiedtherapeutic attributes.

In yet another aspect, the present disclosure provides formulations foruse in methods of treating a kidney disease in a subject in need,comprising: administering to the subject a formulation comprising anadmixture of mammalian renal cells comprising a first cell population,B2, comprising an isolated, enriched population of tubular cells havinga density between 1.045 g/mL and 1.052 g/mL, and a second cellpopulation, B4′, comprising erythropoietin (EPO)-producing cells andvascular cells but depleted of glomerular cells having a density between1.063 g/mL and 1.091 g/mL, wherein the admixture does not include a B1cell population comprising large granular cells of the collecting ductand tubular system having a density of <1.045 g/ml, or a B5 cellpopulation comprising debris and small cells of low granularity andviability with a density >1.091 g/ml. In certain embodiments, the methodincludes determining in a test sample from the subject that the level ofa kidney function indicator is different relative to the indicator levelin a control, wherein the difference in indicator level is indicative ofa reduction in decline, stabilization, or an improvement of one or morekidney functions in the subject. In one embodiment, the B4′ cellpopulation used in the method is characterized by expression of avascular marker. In certain embodiments, the B4′ cell population used inthe method is not characterized by expression of a glomerular marker. Inone embodiment, the admixture of cells used in the method is capable ofoxygen-tunable erythropoietin (EPO) expression. In certain embodiments,the kidney disease to be treated by the methods of the disclosure isaccompanied by an erythropoietin (EPO) deficiency. In certainembodiments, the EPO deficiency is anemia. In some embodiments, the EPOdeficiency or anemia occurs secondary to renal failure in the subject.In some other embodiments, the EPO deficiency or anemia occurs secondaryto a disorder selected from the group consisting of chronic renalfailure, primary EPO deficiency, chemotherapy or anti-viral therapy,non-myeloid cancer, HIV infection, liver disease, cardiac failure,rheumatoid arthritis, or multi-organ system failure. In certainembodiments, the composition used in the method further comprises abiomaterial comprising one or more biocompatible synthetic polymersand/or naturally-occurring proteins or peptides, wherein the admixtureis coated with, deposited on or in, entrapped in, suspended in, embeddedin and/or otherwise combined with the biomaterial. In certainembodiments, the admixture used in the formulations of the disclosure isderived from mammalian kidney tissue or cultured mammalian kidney cells.In other embodiments, the admixture is derived from a kidney sample thatis autologous to the subject in need. In one embodiment, the sample is akidney biopsy. In other embodiments, the formulation contains anadmixture derived from a non-autologous kidney sample.

In yet another aspect, the disclosure provides a use of a formulationcontaining the cell preparations and admixtures described herein or animplantable construct of the instant disclosure for the preparation of amedicament useful in the treatment of a kidney disease, anemia or EPOdeficiency in a subject in need thereof.

In another aspect, the present disclosure provides formulations for usein methods for the regeneration of a native kidney in a subject in needthereof. In one embodiment, the method includes the step ofadministering or implanting a cell population, admixture, or constructdescribed herein to the subject. A regenerated native kidney may becharacterized by a number of indicators including, without limitation,development of function or capacity in the native kidney, improvement offunction or capacity in the native kidney, and the expression of certainmarkers in the native kidney. In one embodiment, the developed orimproved function or capacity may be observed based on the variousindicators of erythroid homeostasis and kidney function described above.In another embodiment, the regenerated kidney is characterized bydifferential expression of one or more stem cell markers. The stem cellmarker may be one or more of the following: SRY (sex determining regionY)-box 2 (Sox2); Undifferentiated Embryonic Cell Transcription Factor(UTF1); Nodal Homolog from Mouse (NODAL); Prominin 1 (PROM1) or CD133(CD133); CD24; and any combination thereof (see Ilagan et al.PCT/US2011/036347 incorporated herein by reference in its entirety). Inanother embodiment, the expression of the stem cell marker(s) isup-regulated compared to a control.

The cell populations described herein, including enriched cellpopulations and admixtures thereof, as well as constructs containing thesame may be used to provide a regenerative effect to a native kidney.The effect may be provided by the cells themselves and/or by productssecreted from the cells. The regenerative effect may be characterized byone or more of the following: a reduction in epithelial-mesenchymaltransition (which may be via attenuation of TGF-β signaling); areduction in renal fibrosis; a reduction in renal inflammation;differential expression of a stem cell marker in the native kidney;migration of implanted cells and/or native cells to a site of renalinjury, e.g., tubular injury, engraftment of implanted cells at a siteof renal injury, e.g., tubular injury; stabilization of one or moreindicators of kidney function (as described herein); restoration oferythroid homeostasis (as described herein); and any combinationthereof.

7. Methods of Monitoring Regeneration

In another aspect, the present disclosure provides a prognostic methodfor monitoring regeneration of a native kidney following administrationor implantation of a formulation containing a cell population,admixture, or construct described herein to the subject. In oneembodiment, the method includes the step of detecting the level ofmarker expression in a test sample obtained from the subject and in acontrol sample, wherein a higher level of expression of the marker inthe test sample, as compared to the control sample, is prognostic forregeneration of the native kidney in the subject. In another embodiment,the method includes the detection of expression of one or more stem cellmarkers in the sample. The stem cell marker may be selected from Sox2;UTF1; NODAL; CD133; CD24; and any combination thereof (see Example 11 ofIlagan et al. PCT/US2011/036347). The detecting step may includedetermining that expression of the stem cell marker(s) is up-regulatedor higher in the test sample relative to a control sample, wherein thehigher level of expression is prognostic for regeneration of thesubject's native kidney. In one other embodiment, mRNA expression of thestem cell marker(s) is detected. In other embodiments, the detection ofmRNA expression may be via a PCR-based method, e.g., qRT-PCR. In situhybridization may also be used for the detection of mRNA expression. Inanother embodiment, polypeptide expression of the stem cell marker mayalso be detected using an anti-stem cell marker agent. In one otherembodiment, the agent is an antibody against the marker. In anotherembodiment, stem cell marker polypeptide expression is detected usingimmunohistochemistry or a Western Blot. Those of ordinary skill in theart will appreciate other methods for detecting mRNA and/or polypeptideexpression of markers.

In another aspect, the disclosure provides methods for prognosticevaluation of a patient following implantation or administration of aformulation containing a cell population, admixture, or constructdescribed herein. In one embodiment, the method includes the step ofdetecting the level of marker expression in a test sample obtained fromsaid subject; (b) determining the expression level in the test samplerelative to the level of marker expression relative to a control sample(or a control reference value); and (c) predicting regenerativeprognosis of the patient based on the determination of marker expressionlevels, wherein a higher level of expression of marker in the testsample, as compared to the control sample (or a control referencevalue), is prognostic for regeneration in the subject.

In one other aspect, the present disclosure provides prognostic methodsfor monitoring regeneration of a native kidney following administrationor implantation of a formulation containing a cell population,admixture, or construct described herein to the subject, in which anon-invasive method is used. As an alternative to a tissue biopsy, aregenerative outcome in the subject receiving treatment can be assessedfrom examination of a bodily fluid, e.g., urine. It has been discoveredthat microvesicles obtained from subject-derived urine sources containcertain components including, without limitation, specific proteins andmiRNAs that are ultimately derived from the renal cell populationsimpacted by treatment with the cell populations of the presentdisclosure. These components may include factors involved in stem cellreplication and differentiation, apoptosis, inflammation andimmuno-modulation. A temporal analysis of microvesicle-associatedmiRNA/protein expression patterns allows for continuous monitoring ofregenerative outcomes within the kidney of subjects receiving the cellpopulations, admixtures, or constructs of the present disclosure.

These kidney-derived vesicles and/or the luminal contents of kidneyderived vesicles shed into the urine of a subject may be analyzed forbiomarkers indicative of regenerative outcome.

In one embodiment, the present disclosure provides methods of assessingwhether a kidney disease (KD) patient is responsive to treatment with atherapeutic formulation. The method may include the step of determiningor detecting the amount of vesicles or their luminal contents in a testsample obtained from a KD patient treated with the therapeutic, ascompared to or relative to the amount of vesicles in a control sample,wherein a higher or lower amount of vesicles or their luminal contentsin the test sample as compared to the amount of vesicles or theirluminal contents in the control sample is indicative of the treatedpatient's responsiveness to treatment with the therapeutic.

The present disclosure also provides a method of monitoring the efficacyof treatment with a therapeutic in a KD patient. In one embodiment, themethod includes the step of determining or detecting the amount ofvesicles in a test sample obtained from a KD patient treated with thetherapeutic, as compared to or relative to the amount of vesicles ortheir luminal contents in a control sample, wherein a higher or loweramount of vesicles or their luminal contents in the test sample ascompared to the amount of vesicles or their luminal contents in thecontrol sample is indicative of the efficacy of treatment with thetherapeutic in the KD patient.

The present disclosure provides a method of identifying a patientsubpopulation for which an agent is effective to treat kidney disease(KD). In one embodiment, the method includes the step of determining acorrelation between efficacy of the agent and the presence of an amountof vesicles or their luminal contents in samples from the patientsubpopulation as compared to the amount of vesicles or their luminalcontents in a sample obtained from a control sample, wherein a higher orlower amount of vesicles in the samples from the patient subpopulationas compared to the amount of vesicles or their luminal contents in thecontrol sample is indicative that the agent is effective to treat KD inthe patient subpopulation.

The determining or detecting step may include analyzing the amount ofmiRNA or other secreted products that may exist in the test sample,e.g., urine.

The non-invasive prognostic methods may include the step of obtaining aurine sample from the subject before and/or after administration orimplantation of a cell population, admixture, or construct describedherein. Vesicles and other secreted products may be isolated from theurine samples using standard techniques including without limitation,centrifugation to remove unwanted debris (Zhou et al. 2008. Kidney Int.74(5):613-621; Skog et al. U.S. Published Patent Application No.20110053157, each of which is incorporated herein by reference in itsentirety).

The present disclosure relates to non-invasive methods to detectregenerative outcome in a subject following treatment. The methodsinvolve detection of vesicles or their luminal contents in urine from atreated subject. The luminal contents may be one or more miRNAs. Thedetection of combinations or panels of the individual miRNAs may besuitable for such prognostic methods. Exemplary combinations include twoor more of the following: miR-24; miR-195; miR-871; miR-30b-5p; miR-19b;miR-99a; miR-429; let-7f; miR-200a; miR-324-5p; miR-10a-5p; and anycombination thereof. In one embodiment, the combination of miRNAs mayinclude 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more individual miRNAs. Thoseof ordinary skill in the art will appreciate that other miRNAs andcombinations of miRNAs may be suitable for use in such prognosticmethods. Sources of additional miRNAs include miRBase athttp://mirbase.org, which is hosted and maintained in the Faculty ofLife Sciences at the University of Manchester.

Those of skill in the art will appreciate that the prognostic methodsfor detecting regeneration may be suitable for subjects treated withother therapeutics known in the art, apart from the cell populations andconstructs described herein.

In some embodiments, the determining step comprises the use of asoftware program executed by a suitable processor for the purpose of (i)measuring the differential level of marker expression (orvesicles/vesicle contents) in a test sample and a control; and/or (ii)analyzing the data obtained from measuring differential level of markerexpression in a test sample and a control. Suitable software andprocessors are well known in the art and are commercially available. Theprogram may be embodied in software stored on a tangible medium such asCD-ROM, a floppy disk, a hard drive, a DVD, or a memory associated withthe processor, but persons of ordinary skill in the art will readilyappreciate that the entire program or parts thereof could alternativelybe executed by a device other than a processor, and/or embodied infirmware and/or dedicated hardware in a well known manner.

Following the determining step, the measurement results, findings,diagnoses, predictions and/or treatment recommendations are typicallyrecorded and communicated to technicians, physicians and/or patients,for example. In certain embodiments, computers will be used tocommunicate such information to interested parties, such as, patientsand/or the attending physicians. In some embodiments, the assays will beperformed or the assay results analyzed in a country or jurisdictionwhich differs from the country or jurisdiction to which the results ordiagnoses are communicated.

In a preferred embodiment, a prognosis, prediction and/or treatmentrecommendation based on the level of marker expression measured in atest subject having a differential level of marker expression iscommunicated to the subject as soon as possible after the assay iscompleted and the prognosis and/or prediction is generated. The resultsand/or related information may be communicated to the subject by thesubject's treating physician. Alternatively, the results may becommunicated directly to a test subject by any means of communication,including writing, electronic forms of communication, such as email, ortelephone. Communication may be facilitated by use of a computer, suchas in case of email communications. In certain embodiments, thecommunication containing results of a prognostic test and/or conclusionsdrawn from and/or treatment recommendations based on the test, may begenerated and delivered automatically to the subject using a combinationof computer hardware and software which will be familiar to artisansskilled in telecommunications. One example of a healthcare-orientedcommunications system is described in U.S. Pat. No. 6,283,761; however,the present disclosure is not limited to methods which utilize thisparticular communications system. In certain embodiments of the methodsof the disclosure, all or some of the method steps, including theassaying of samples, prognosis and/or prediction of regeneration, andcommunicating of assay results or prognoses, may be carried out indiverse (e.g., foreign) jurisdictions.

In another aspect, the prognostic methods described herein provideinformation to an interested party concerning the regenerative successof the implantation or administration.

In all embodiments, the methods of providing a regenerated kidney to asubject in need of such treatment as described herein may include thepost-implantation step of prognostic evaluation of regeneration asdescribed above.

8. Bioactive Cell Formulations

The formulations described herein incorporate biomaterials havingproperties which create a favorable environment for the active agent,such as bioactive renal cells, to be administered to a subject. In oneembodiment, the formulation contains a first biomaterial that provides afavorable environment from the time the active agent is formulated withthe biomaterial up until the point of administration to the subject. Inone other embodiment, the favorable environment concerns the advantagesof having bioactive cells suspended in a substantially solid stateversus cells in a fluid (as described herein) prior to administration toa subject. In another embodiment, the first biomaterial is atemperature-sensitive biomaterial. The temperature-sensitive biomaterialmay have (i) a substantially solid state at about 8° C. or below, and(ii) a substantially liquid state at ambient temperature or above. Inone embodiment, the ambient temperature is about room temperature.

In another aspect, the formulation contains bioactive cells combinedwith a second biomaterial that provides a favorable environment for thecombined cells from the time of formulation up until a point afteradministration to the subject. In one embodiment, the favorableenvironment provided by the second biomaterial concerns the advantagesof administering cells in a biomaterial that retains structuralintegrity up until the point of administration to a subject and for aperiod of time after administration. In one embodiment, the structuralintegrity of the second biomaterial following implantation is minutes,hours, days, or weeks. In one embodiment, the structural integrity isless than one month, less than one week, less than one day, or less thanone hour. The relatively short term structural integrity provides aformulation that can deliver the active agent and biomaterial to atarget location in a tissue or organ with controlled handling, placementor dispersion without being a hindrance or barrier to the interaction ofthe incorporated elements with the tissue or organ into which it wasplaced.

In another embodiment, the second biomaterial is a temperature-sensitivebiomaterial that has a different sensitivity than the first biomaterial.The second biomaterial may have (i) a substantially solid state at aboutambient temperature or below, and (ii) a substantially liquid state atabout 37° C. or above. In one embodiment, the ambient temperature isabout room temperature.

In one embodiment, the second biomaterial is crosslinked beads. Thecrosslinked beads may have finely tunable in vivo residence timesdepending on the degree of crosslinking, as described herein. In anotherembodiment, the crosslinked beads comprise bioactive cells and areresistant to enzymatic degradation as described herein.

The formulations of the present disclosure may include the firstbiomaterial combined with an active agent, e.g., bioactive cells, withor without a second biomaterial combined with an active agent, e.g.,bioactive cells. Where a formulation includes a second biomaterial, itmay be a temperature sensitive bead and/or a crosslinked bead. Variousrepresentative formulations are provided in the examples below (see alsoFIGS. 3-7).

The bioactive cell preparations, admixtures, and/or constructs describedherein can be administered as bioactive cell formulations. In oneaspect, the formulations include the cells and one or more biomaterialsthat provide stability to the bioactive cell preparations, admixtures,and/or constructs described herein. In one embodiment, the biomaterialis a temperature-sensitive biomaterial that can maintain at least twodifferent phases or states depending on temperature. The biomaterial iscapable of maintaining a first state at a first temperature, a secondstate at a second temperature, and/or a third state at a thirdtemperature. The first, second or third state may be a substantiallysolid, a substantially liquid, or a substantially semi-solid orsemi-liquid state. In one embodiment, the biomaterial has a first stateat a first temperature and a second state at a second temperature,wherein the first temperature is lower than the second temperature.

In one other embodiment, the state of the temperature-sensitivebiomaterial is a substantially solid state at a temperature of about 8°C. or below. In other embodiments, the substantially solid state ismaintained at about 1° C., about 2° C., about 3° C., about 4° C., about5° C., about 6° C., about 7° C., or about 8° C. In one embodiment, thesubstantially solid state has the form of a gel. In other embodiments,the state of the temperature-sensitive biomaterial is a substantiallyliquid state at ambient temperature or above. In one embodiment, thesubstantially liquid state is maintained at about 31° C., about 32° C.,about 33° C., about 34° C., about 35° C., about 36° C., or about 37° C.In one embodiment, the ambient temperature is about room temperature.

In another embodiment, the state of the temperature-sensitivebiomaterial is a substantially solid state at a temperature of aboutambient temperature or below. In one embodiment, the ambient temperatureis about room temperature. In another embodiment, the substantiallysolid state is maintained at about 17° C., about 16° C., about 15° C.,about 14° C., about 13° C., about 12° C., about 11° C., about 10° C.,about 9° C., about 8° C., about 7° C., about 6° C., about 5° C., about4° C., about 3° C., about 2° C., or about 1° C. In one embodiment, thesubstantially solid state has the form of a bead. In another embodiment,the state of the temperature-sensitive biomaterial is a substantiallyliquid state at a temperature of about 37° C. or above. In one otherembodiment, the substantially solid state is maintained at about 37° C.,about 38° C., about 39° C., or about 40° C.

The temperature-sensitive biomaterials may be provided in the form of asolution, in the form of beads, or in other suitable forms describedherein and/or known to those of ordinary skill in the art. The cellpopulations and preparations described herein may be coated with,deposited on, embedded in, attached to, seeded, suspended in, orentrapped in a temperature-sensitive biomaterial. Alternatively, thetemperature-sensitive biomaterial may be provided without any cells,such as, for example in the form of spacer beads.

In other embodiments, the temperature-sensitive biomaterial has atransitional state between a first state and a second state. In oneembodiment, the transitional state is a solid-to-liquid transitionalstate between a temperature of about 8° C. and about ambienttemperature. In one embodiment, the ambient temperature is about roomtemperature. In one other embodiment, the solid-to-liquid transitionalstate occurs at one or more temperatures of about 8° C., about 9° C.,about 10° C., about 11° C., about 12° C., about 13° C., about 14° C.,about 15° C., about 16° C., about 17° C., and about 18° C.

The temperature-sensitive biomaterials have a certain viscosity at agiven temperature measured in centipoise (cP). In one embodiment, thebiomaterial has a viscosity at 25° C. of about 1 cP to about 5 cP, about1.1 cP to about 4.5 cP, about 1.2 cP to about 4 cP, about 1.3 cP toabout 3.5 cP, about 1.4 cP to about 3.5 cP, about 1.5 cP to about 3 cP,about 1.55 cP to about 2.5 cP, or about 1.6 cP to about 2 cP. In anotherembodiment, the 0.75% (w/v) solution has a viscosity at 37° C. of about1.0 cP to about 1.15 cP. The viscosity at 37° C. may be about 1.0 cP,about 1.01 cP, about 1.02 cP, about 1.03 cP, about 1.04 cP, about 1.05cP, about 1.06 cP, about 1.07 cP, about 1.08 cP, about 1.09 cP, about1.10 cP, about 1.11 cP, about 1.12 cP, about 1.13 cP, about 1.14 cP, orabout 1.15 cP. In one other embodiment, the biomaterial is a gelatinsolution. The gelatin is present at about 0.5%, about 0.55%, about 0.6%,about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about0.9%, about 0.95% or about 1%, (w/v) in the solution. In one example,the biomaterial is a 0.75% (w/v) gelatin solution in PBS. In oneembodiment, the 0.75% (w/v) solution has a viscosity at 25° C. of about1.6 cP to about 2 cP. In one embodiment, the 0.75% (w/v) solution has aviscosity at 37° C. of about 1.07 cP to about 1.08 cP. The gelatinsolution may be provided in PBS, DMEM, or another suitable solvent.

In one aspect, the bioactive cell formulation also includes a cellviability agent. In one embodiment, the cell viability agent is selectedfrom the group consisting of an antioxidant, an oxygen carrier, animmunomodulatory factor, a cell recruitment factor, a cell attachmentfactor, an anti-inflammatory agent, an angiogenic factor, a woundhealing factor, and products secreted from bioactive cells.

Antioxidants are characterized by the ability to inhibit oxidation ofother molecules. Antioxidants include, without limitation, one or moreof 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®),carotenoids, flavonoids, isoflavones, ubiquinone, glutathione, lipoicacid, superoxide dismutase, ascorbic acid, vitamin E, vitamin A, mixedcarotenoids (e.g., beta carotene, alpha carotene, gamma carotene,lutein, lycopene, phytopene, phytofluene, and astaxanthin), selenium,Coenzyme Q10, indole-3-carbinol, proanthocyanidins, resveratrol,quercetin, catechins, salicylic acid, curcumin, bilirubin, oxalic acid,phytic acid, lipoic acid, vanilic acid, polyphenols, ferulic acid,theaflavins, and derivatives thereof. Those of ordinary skill in the artwill appreciate other suitable antioxidants for use in the presentdisclosure.

Oxygen carriers are agents characterized by the ability to carry andrelease oxygen. They include, without limitation, perfluorocarbons andpharmaceuticals containing perfluorocarbons. Suitableperfluorocarbon-based oxygen carriers include, without limitation,perfluorooctyl bromide (C8F17Br); perfluorodichorotane (C8F16C12);perfluorodecyl bromide; perfluobron; perfluorodecalin;perfluorotripopylamine; perfluoromethylcyclopiperidine; Fluosol®(perfluorodecalin & perfluorotripopylamine); Perftoran®(perfluorodecalin & perfluoromethylcyclopiperidine); Oxygent®(perfluorodecyl bromide & perfluobron); Ocycyte™ (perfluoro(tert-butylcyclohexane)). Those of ordinary skill in the art willappreciate other suitable perfluorocarbon-based oxygen carriers for usein the present disclosure.

Immunomodulatory factors include, without limitation, osteopontin, FASLigand factors, interleukins, transforming growth factor beta, plateletderived growth factor, clusterin, transferrin, regulated upon action,normal T-cell expressed, secreted protein (RANTES), plasminogenactivator inhibitor-1 (Pai-1), tumor necrosis factor alpha (TNF-alpha),interleukin 6 (IL-6), alpha-1 microglobulin, and beta-2-microglobulin.Those of ordinary skill in the art will appreciate other suitableimmunomodulatory factors for use in the present disclosure.

Anti-inflammatory agents or immunosuppressant agents (described below)may also be part of the formulation. Those of ordinary skill in the artwill appreciate other suitable antioxidants for use in the presentformulations and/or treatments.

Cell recruitment factors include, without limitation, monocytechemotatic protein 1 (MCP-1), and CXCL-1. Those of ordinary skill in theart will appreciate other suitable cell recruitment factors for use inthe present formulations and/or treatments.

Cell attachment factors include, without limitation, fibronectin,procollagen, collagen, ICAM-1, connective tissue growth factor,laminins, proteoglycans, specific cell adhesion peptides such as RGD andYSIGR. Those of ordinary skill in the art will appreciate other suitablecell attachment factors for use in the present formulations and/ortreatments.

Anglogenic factors include, without limitation, matrix metalloprotease 1(MMP1), matrix metalloprotease 2 (MMP2), vascular endothelial growthfactor F (VEGF), matrix metalloprotease 9 (MMP-9), tissue inhibitor ormatalloproteases-1 (TIMP-1) vascular endothelial growth factor F (VEGF),angiopoietin-2 (ANG-2). Those of ordinary skill in the art willappreciate other suitable angiogenic factors for use in the presentformulations and/or treatments.

Wound healing factors include, without limitation, keratinocyte growthfactor 1 (KGF-1), tissue plasminogen activator (tPA), calbindin,clusterin, cystatin C, trefoil factor 3. Those of ordinary skill in theart will appreciate other suitable wound healing factors for use in thepresent formulations and/or treatments.

Secreted products from bioactive cells described herein may also beadded to the bioactive cell formulation as a cell viability agent.

In one other aspect, the formulation includes a temperature-sensitivebiomaterial described herein and a population of biocompatible beadscontaining a biomaterial. In one embodiment, the beads are crosslinked.Crosslinking may be achieved using any suitable crosslinking agent knownto those of ordinary skill in the art, such as, for example,carbodiimides; aldehydes (e.g. furfural, acrolein, formaldehyde,glutaraldehyde, glyceryl aldehyde), succinimide-based crosslinkers{Bis(sulfosuccinimidyl) suberate (BS3), Disuccinimidyl glutarate (DSG),Disuccinimidyl suberate (DSS), Dithiobis(succinimidyl propionate),Ethylene glycolbis(sulfosuccinimidylsuccinate), Ethyleneglycolbis(succinimidylsuccinate) (EGS), Bis(Sulfosuccinimidyl) glutarate(BS2G), Disuccinimidyl tartrate (DST)); epoxides (Ethylene glycoldiglycidyl ether, 1,4 Butanediol diglycidyl ether); saccharides (glucoseand aldose sugars); sulfonic acids and p-toluene sulfonic acid;carbonyldlimidazole; genipin; imines; ketones; diphenylphosphorylazide(DDPA); terephthaloyl chloride; cerium (III) nitrate hexahydrate;microbial transglutaminase; and hydrogen peroxide. Those of ordinaryskill in the art will appreciate other suitable crosslinking agents andcrosslinking methods for use in the present methods, formulations and/ortreatments.

In one embodiment, the beads are carbodlimide-crosslinked beads. Thecarbodiimide-crosslinked beads may be crosslinked with a carbodiimideselected from the group consisting of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), DCC—N,N′-dicyclohexylcarbodiimide(DCC), and N,N′-Diisopropylcarbodiimide (DIPC). Beads treated with lowerconcentration of EDC were expected to have a higher number of freeprimary amines, while samples treated with high concentrations ofcrosslinker would have most of the primary amines engaged in amidebonds. The intensity of the orange color developed by the covalentbonding between the primary amine and picrylsulfonic acid, detectablespectrophotometrically at 335 nm, is proportional to the number ofprimary amines present in the sample. When normalized per milligram ofprotein present in the sample, an inverse correlation between the numberof free amines present and the initial concentration of EDC used forcrosslinking can be observed. This result is indicative of differentialbead crosslinking, dictated by the amount of carbodiimide used in thereaction. In general, crosslinked beads exhibit a reduced number of freeprimary amines as compared to non-crosslinked beads. The number of freeprimary amines may be detected spectrophotometrically at about 335 nm.

The crosslinked beads have a reduced susceptibility to enzymaticdegradation as compared to non-crosslinked biocompatible beads, therebyproviding beads with finely tunable in vivo residence times. Forexample, the cross-linked beads are resistant to endogenous enzymes,such as collagenases. The provision of crosslinked beads is part of adelivery system focused on the development and production ofbiomaterials that facilitate one or more of: (a) delivery of attachedcells to the desired sites and creation of space for regeneration andingrowth of native tissue and vascular supply; (b) ability to persist atthe site long enough to allow cells to establish, function, remodeltheir microenvironment and secrete their own extracellular matrix (ECM);(c) promotion of integration of the transplanted cells with thesurrounding tissue; (d) ability to implant cells in a substantiallysolid form; (e) short term structural integrity that does not provide asignificant barrier to tissue ingrowth or integration of deliveredcells/materials with the host tissue; (f) localized in vivo delivery ina substantially solid form thereby preventing dispersion of cells withinthe tissue during implantation; (g) improved stability and viability ofanchorage dependent cells compared to cells suspended in a fluid; and(h) biphasic release profile when cells are delivered i) in asubstantially solid form (e.g., attached to beads), and ii) in asubstantially liquid form (e.g., suspended in a fluid).

In one embodiment, the present disclosure provides crosslinked beadscontaining gelatin. Non-crosslinked gelatin beads are not suitable for abioactive cell formulation because they rapidly lose integrity and cellsdissipate from the injection site. In contrast, highly crosslinkedgelatin beads may persist too long at the injection site and may hinderthe de-novo ECM secretion, cell integration and tissue regeneration. Thepresent disclosure allows for the in vivo residence time of thecrosslinked beads to be finely tuned. In order to tailor thebiodegradability of biomaterials, different crosslinker concentrationsof carbodiimide are used while the overall reaction conditions were keptconstant for all samples. For example, the enzymatic susceptibility ofcarbodiimide-crosslinked beads can be finely tuned by varying theconcentration of crosslinking agent from about zero to about 1M. In someembodiments, the concentration is about 5 mM, about 6 mM, about 7 mM,about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM,about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM,about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM,about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 55 mM,about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about85 mM, about 90 mM, about 95 mM, or about 100 mM. The crosslinkerconcentration may also be about 0.15 M, about 0.2 M, about 0.25 M, about0.3 M, about 0.35 M, about 0.4 M, about 0.45 M, about 0.5 M, about 0.55M, about 0.6 M, about 0.65 M, about 0.7 M, about 0.75 M, about 0.8 M,about 0.85 M, about 0.9 M, about 0.95 M, or about 1 M. In anotherembodiment, the crosslinking agent is 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). In one embodiment, the EDC-crosslinkedbeads are gelatin beads.

Cross-linked beads may have certain characteristics that favor theseeding, attachment, or encapsulation. For example, the beads may have aporous surface and/or may be substantially hollow. The presence of poresprovides an increased cell attachment surface allowing for a greaternumber of cells to attach as compared to a non-porous or smooth surface.In addition, the pore structure can support host tissue integration withthe porous beads supporting the formation of de novo tissue. The beadshave a size distribution that can be fitted to a Weibull plotcorresponding to the general particle distribution pattern. In oneembodiment, the cross-linked beads have an average diameter of less thanabout 120 μm, about 115 μm, about 110 μm, about 109 μm, about 108 μm,about 107 μm, about 106 μm, about 105 μm, about 104 μm, about 103 μm,about 102 μm, about 101 μm, about 100 μm, about 99 μm, about 98 μm,about 97 μm, about 96 μm, about 95 μm, about 94 μm, about 93 μm, about92 μm, about 91 μm, or about 90 μm. The characteristics of thecross-linked beads vary depending upon the casting process. Forinstance, a process in which a stream of air is used to aerosolize aliquid gelatin solution and spray it into liquid nitrogen with a thinlayer chromatography reagent sprayer (ACE Glassware) is used to providebeads having the aforementioned characteristics. Those of skill in theart will appreciate that modulating the parameters of the castingprocess provides the opportunity to tailor different characteristics ofthe beads, e.g., different size distributions.

The cytocompatibility of the cross-linked beads is assessed in vitroprior to formulation using cell culture techniques in which beads arecultured with cells that correspond to the final bioactive cellformulation. For instance, the beads are cultured with primary renalcells prior to preparation of a bioactive renal cell formulation andlive/dead cell assays are used to confirm cytocompatibility. In certainformulations, the biocompatible cross-linked beads are combined with atemperature-sensitive biomaterial in solution at about 5% (w/w) to about15% (w/w) of the volume of the solution. The cross-linked beads may bepresent at about 5% (w/w), about 5.5% (w/w), about 6% (w/w), about 6.5%(w/w), about 7% (w/w), about 7.5% (w/w), about 8% (w/w), about 8.5%(w/w), about 9% (w/w), about 9.5% (w/w), about 10% (w/w), about 10.5%(w/w), about 11% (w/w), about 11.5% (w/w), about 12% (w/w), about 12.5%(w/w), about 13% (w/w), about 13.5% (w/w), about 14% (w/w), about 14.5%(w/w), or about 15% (w/w) of the volume of the solution.

In another aspect, the present disclosure provides formulations thatcontain biomaterials which degrade over a period time on the order ofminutes, hours, or days. This is in contrast to a large body or workfocusing on the implantation of solid materials that then slowly degradeover days, weeks, or months.

In another aspect, the present disclosure provides formulations havingbiocompatible cross-linked beads seeded with bioactive cells togetherwith a delivery matrix. In one embodiment, the delivery matrix has oneor more of the following characteristics: biocompatibility,biodegradable/bioresorbable, a substantially solid state prior to andduring implantation into a subject, loss of structural integrity(substantially solid state) after implantation, and cytocompatibleenvironment to support cellular viability. The delivery matrix's abilityto keep implanted particles (e.g., crosslinked beads) spaced out duringimplantation enhances native tissue ingrowth. If the delivery matrix isabsent, then compaction of cellularized beads during implantation canlead to inadequate room for sufficient tissue ingrowth. The deliverymatrix facilitates implantation of solid formulations. In addition, theshort duration of the structural integrity means that soon afterimplantation, the matrix does not provide a significant barrier totissue ingrowth or integration of the delivered cells/materials withhost tissue. The delivery matrix provides for localization of theformulation described herein since inserted of a solid unit helpsprevent the delivered materials from dispersing within the tissue duringimplantation. For cell-based formulations, a solid delivery matriximproves stability and viability of anchorage dependent cells comparedto cells suspended in a fluid.

In one embodiment, the delivery matrix is a population of biocompatiblebeads that is not seeded with cells. In another embodiment, the unseededbeads are dispersed throughout and in between the individual cell-seededbeads. The unseeded beads act as “spacer beads” between the cell-seededbeads prior to and immediately after transplantation. The spacer beadscontain a temperature-sensitive biomaterial having a substantially solidstate at a first temperature and a substantially liquid state at asecond temperature, wherein the first temperature is lower than thesecond temperature. For example, the spacer beads contain a biomaterialhaving a substantially solid state at about ambient temperature or belowand a substantially liquid state at about 37° C., such as that describedherein. In one embodiment, the ambient temperature is about roomtemperature. In another embodiment, the biomaterial is a gelatinsolution. The gelatin solution is present at about 4%, about 4.5%, about5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%,about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, or about 11%,(w/v). The gelatin solution may be provided in PBS, cell culture media(e.g., DMEM), or another suitable solvent.

In one aspect, the present disclosure provides formulations that containbiomaterials which are implanted in a substantially solid form (e.g.,spacer beads) and then liquefy/melt or otherwise lose structuralintegrity following implantation into the body. This is in contrast tothe significant body of work focusing on the use of materials that canbe injected as a liquid, which then solidify in the body.

The temperature-sensitivity of spacer beads can be assessed in vitroprior to formulation. Spacer beads can be labeled and mixed withunlabeled non-temperature-sensitive beads. The mixture is then incubatedat 37° C. to observe changes in physical transition. The loss of shapeof the labeled temperature-sensitive beads at the higher temperature isobserved over time. For example, temperature-sensitive gelatin beads maybe made with Alcian blue dye to serve as a marker of physicaltransition. The blue gelatin beads are mixed with Cultispher S beads(white), loaded into a catheter, then extruded and incubated in IX PBS,pH 7.4, at 37° C. The loss of shape of the blue gelatin beads isfollowed microscopically at different time points. Changes in thephysical state of the blue gelatin beads are visible after 30 minbecoming more pronounced with prolonged incubation times. The beads donot completely dissipate because of the viscosity of the material.

The bioactive cell formulations described herein may be used to preparerenal cell-based formulations for injection into the kidney. However,those of ordinary skill in the art will appreciate that the formulationswill be suitable for many other types of bioactive cell populations. Forexample, the present disclosure contemplates formulations for bioactivecells for injection into any solid organ or tissue.

In one aspect, the bioactive cell formulations described herein willcontain a set number of cells. In one embodiment, the total number ofcells for the formulation is about 10⁴, about 10⁵, about 10⁶, about 10⁷,about 10⁸, or about 10⁹. In one embodiment, the dosage of cells for aformulation described herein may be calculated based on the estimatedmass or functional mass of the target organ or tissue. In certainembodiments, the bioactive cell formulations contain a dosagecorresponding to a number of cells based upon the weight of the hostorgan that will be the subject of treatment by the formulation. Forexample, a bioactive renal cell formulation is based upon an averageweight of about 150 grams for a human kidney. In one embodiment, thenumber of cells per gram (g) of kidney is about 600 cells/g to about7.0×10⁷ cells/g. In some embodiments, the number of cells per gram ofkidney is about 600 cells/g, about 1000 cells/g, about 1500 cells/g,about 2000 cells/g, about 2500 cells/g, about 3000 cells/g, about 3500cells/g, about 4000 cells/g, about 4500 cells/g, about 5000 cells/g,about 5500 cells/g, about 6000 cells/g, about 6500 cells/g, about 7000cells/g, about 7500 cells/g, about 8000 cells/g, about 8500 cells/g,about 9000 cells/g, about 9500 cells/g, or about 10,000 cells/g.

In other embodiments, the number of cells per gram of kidney is about1.5×10⁴ cells/g, about 2.0×10⁴ cells/g, about 2.5×10⁴ cells/g, about3.0×10⁴ cells/g, about 3.5×10⁴ cells/g, about 4.0×10⁴ cells/g, about4.5×10⁴ cells/g, about 5.0×10⁴ cells/g, about 5.5×10⁴ cells/g, about6.0×10⁴ cells/g, about 6.5×10⁴ cells/g, about 7.0×10⁴ cells/g, about7.5×10⁴ cells/g, about 8.0×10⁴ cells/g, about 9.5×10⁴ cells/g.

In other embodiments, the number of cells per gram of kidney is about1.0×10⁵ cells/g, about 1.5×10⁵ cells/g, about 2.0×10⁵ cells/g, about2.5×10⁵ cells/g, about 3.0×10⁵ cells/g, about 3.5×10⁵ cells/g, about4.0×10⁵ cells/g, about 4.5×10⁵ cells/g, about 5.0×10⁵ cells/g, about5.5×10⁵ cells/g, about 6.0×10⁵ cells/g, about 6.5×10⁵ cells/g, about7.0×10⁵ cells/g, about 7.5×10⁵ cells/g, about 8.0×10⁵ cells/g, about8.5×10⁵ cells/g, about 9.0×10⁵ cells/g, or about 9.5×10⁵ cells/g.

In other embodiments, the number of cells per gram of kidney is about1.0×10⁶ cells/g, about 1.5×10⁶ cells/g, about 2.0×10⁶ cells/g, about2.5×10⁶ cells/g, about 3.0×10⁶ cells/g, about 3.5×10⁶ cells/g, about4.0×10⁶ cells/g, about 4.5×10⁶ cells/g, about 5.0×10⁶ cells/g, about5.5×10⁶ cells/g, about 6.0×10⁶ cells/g, about 6.5×10⁶ cells/g, about7.0×10⁶ cells/g, about 7.5×10⁶ cells/g, about 8.0×10⁶ cells/g about8.5×10⁶ cells/g, about 9.0×10⁶ cells/g, about 9.5×10⁶ cells/g, 1.0×10⁷cells/g, or about 1.5×10⁷ cells/g.

A total number of cells may be selected for the formulation and thevolume of the formulation may be adjusted to reach the proper dosage.

In some embodiments, the formulation may contain a dosage of cells to asubject that is a single dosage or a single dosage plus additionaldosages. In other embodiments, the dosages may be provided by way of aconstruct as described herein. The therapeutically effective amount ofthe renal cell populations or admixtures of renal cell populationsdescribed herein can range from the maximum number of cells that issafely received by the subject to the minimum number of cells necessaryfor treatment of kidney disease, e.g., stabilization, reducedrate-of-decline, or improvement of one or more kidney functions.

The therapeutically effective amount of the renal cell populations oradmixtures thereof described herein can be suspended in apharmaceutically acceptable carrier or excipient. Such a carrierincludes, but is not limited to basal culture medium plus 1% serumalbumin, saline, buffered saline, dextrose, water, collagen, alginate,hyaluronic acid, fibrin glue, polyethyleneglycol, polyvinylalcohol,carboxymethylcellulose and combinations thereof. The formulation shouldsuit the mode of administration.

Accordingly, the disclosure provides a use of a formulation containingrenal cell populations or admixtures thereof, for example, the B2 cellpopulation alone or admixed with the B3 and/or B4 or B4′ cellpopulation, for the manufacture of a medicament to treat kidney diseasein a subject. In some embodiments, the medicament further comprisesrecombinant polypeptides, such as growth factors, chemokines orcytokines. In further embodiments, the medicaments comprise a humankidney-derived cell population. The cells used to manufacture themedicaments can be isolated, derived, or enriched using any of thevariations provided for the methods described herein.

The renal cell preparation(s), or admixtures thereof, or compositionsare formulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for administration to human beings. Typically,compositions for intravenous administration, intra-arterialadministration or administration within the kidney capsule, for example,are solutions in sterile isotonic aqueous buffer. Where necessary, thecomposition can also include a local anesthetic to ameliorate any painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa cryopreserved concentrate in a hermetically sealed container such asan ampoule indicating the quantity of active agent. When the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientscan be mixed prior to administration.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions (see,e.g., Alfonso R Gennaro (ed), Remington: The Science and Practice ofPharmacy, formerly Remington's Pharmaceutical Sciences 20th ed.,Uppincott, Williams & Wilkins, 2003, incorporated herein by reference inits entirety). The pharmaceutical compositions are generally formulatedas sterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

One aspect further provides a pharmaceutical formulation, comprising arenal cell preparation, for example, the B2 cell preparation alone orincombination with the B3 and/or B4 or B4′ cell preparation, and apharmaceutically acceptable carrier. In some embodiments, theformulation comprises from 10⁴ to 10⁶ mammalian kidney-derived cells.

Modified Release Formulations

In one aspect, the formulations of the present disclosure are providedas modified release formulations. In general, the modified release ischaracterized by an initial release of a first active agent uponadministration following by at least one additional, subsequent releaseof a second active agent. The first and second active agents may be thesame or they may be different. In one embodiment, the formulationsprovide modified release through multiple components in the sameformulation. In another embodiment, the modified release formulationcontains an active agent as part of a first component that allows theactive agent to move freely throughout the volume of the formulation,thereby permitting immediate release at the target site uponadministration. The first component may be a temperature-sensitivebiomaterial having a substantially liquid phase and a substantiallysolid phase, wherein the first component is in a substantially liquidphase at the time of administration. In one embodiment, the active agentin the substantially liquid phase such that it is substantially free tomove throughout the volume of the formulation, and therefore isimmediately released to the target site upon administration.

In another embodiment, the modified release formulation has an activeagent as part of a second component in which the active agent isattached to, deposited on, coated with, embedded in, seeded upon, orentrapped in the second component, which persists before and afteradministration to the target site. The second component containsstructural elements with which the active agent is able to associatewith, thereby preventing immediate release of the active agent from thesecond component at the time of administration. For example, the secondcomponent is provided in a substantially solid form, e.g., biocompatiblebeads, which may be crosslinked to prevent or delay in vivo enzymaticdegradation. In one embodiment, the active agent in the substantiallysolid phase retains its structural integrity within the formulationbefore and after administration and therefore it does not immediatelyrelease the active agent to the target site upon administration.Suitable carriers for modified release formulations have been describedherein but those of ordinary skill in the art will appreciate othercarriers that are appropriate for use herein.

In one embodiment, the formulation provides an initial rapiddelivery/release of delivered elements, including cells, nanoparticles,therapeutic molecules, etc. followed by a later delayed release ofelements. The formulations of the present disclosure can be designed forsuch biphasic release profile where the agent to be delivered isprovided in both an unattached form (e.g., cells in a solution) and anattached form (e.g., cells together with beads or another suitablecarrier). Upon initial administration, the unencumbered agent isprovided immediately to the site of delivery while release of theencumbered agent is delayed until structural integrity of the carrier(e.g., beads) fails at which point the previously attached agent isreleased. As discussed below, other suitable mechanisms of release willbe appreciated by those of ordinary skill in the art.

The time delay for release can be adjusted based upon the nature of theactive agent. For example, the time delay for release in a bioactivecell formulation may be on the order of seconds, minutes, hours, ordays. In some circumstances, a delay on the order of weeks may beappropriate. For other active agents, such as small or large molecules,the time delay for release in a formulation may be on the order ofseconds, minutes, hours, days, weeks, or months. It is also possible forthe formulation to contain different biomaterials that provide differenttime delay release profiles. For example, a first biomaterial with afirst active agent may have a first release time and a secondbiomaterial with a second active agent may have a second release time.The first and second active agent may be the same or different.

As discussed herein, the time period of delayed release may generallycorrespond to the time period for loss of structural integrity of abiomaterial. However, those of ordinary skill in the art will appreciateother mechanisms of delayed release. For example, an active agent may becontinually released over time independent of the degradation time ofany particular biomaterial, e.g., diffusion of a drug from a polymericmatrix. In addition, bioactive cells can migrate away from a formulationcontaining a biomaterial and the bioactive cells to native tissue. Inone embodiment, bioactive cells migrate off of a biomaterial, e.g., abead, to the native tissue.

Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Prolonged absorption of injectableformulations can be brought about by including in the formulation anagent that delays absorption, for example, monostearate salts andgelatin. Many methods for the preparation of such formulations arepatented or generally known to those skilled in the art. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978. Additional methods applicableto the controlled or extended release of polypeptide agents aredescribed, for example, in U.S. Pat. Nos. 6,306,406 and 6,346,274, aswell as, for example, in U.S. Patent Application Nos. US20020182254 andUS20020051808, all of which are incorporated herein by reference.

9. Methods and Routes of Administration

The bioactive cell formulations of the present disclosure can beadministered alone or in combination with other bioactive components.The formulations are suitable for injection or implantation ofincorporated tissue engineering elements to the interior of solid organsto regenerate tissue. In addition, the formulations are used for theinjection or implantation of tissue engineering elements to the wall ofhollow organs to regenerate tissue.

In one aspect, the present disclosure provides methods of providing abioactive cell formulation described herein to a subject in need. In oneembodiment, the source of the bioactive cell may be autologous orallogeneic, syngeneic (autogeneic or isogeneic), and any combinationthereof. In instances where the source is not autologous, the methodsmay include the administration of an immunosuppressant agent. Suitableimmunosuppressant drugs include, without limitation, azathioprine,cyclophosphamide, mizoribine, ciclosporin, tacrolimus hydrate,chlorambucil, Iobenzarit disodium, auranofin, alprostadil, gusperimushydrochloride, biosynsorb, muromonab, alefacept, pentostatin,daclizumab, sirolimus, mycophenolate mofetil, leflonomide, basiliximab,dornase a, bindarid, cladribine, pimecrolimus, ilodecakin, cedelizumab,efalizumab, everolimus, anisperimus, gavilimomab, faralimomab,clofarabine, rapamycin, siplizumab, saireito, LDP-03, CD4, SR-43551,SK&F-106615, IDEC-114, IDEC-131, FTY-720, TSK-204, LF-080299, A-86281,A-802715, GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-1011, MT-1345,CNI-1493, CBP-2011, J-695, UP-920, L-732531, ABX-RB2, AP-1903, IDPS,BMS-205820, BMS-224818, CTLA4-1g, ER-49890, ER-38925, ISAtx-247, RDP-58,PNU-156804, UP-1082, TMC-95A, TV-4710, PTR-262-MG, and AGI-1096 (seeU.S. Pat. No. 7,563,822). Those of ordinary skill in the art willappreciate other suitable immunosuppressant drugs.

The treatment methods of the subject disclosure involve the delivery ofa bioactive cell formulation described herein. In one embodiment, directadministration of cells to the site of intended benefit is preferred. Asubject in need may also be treated by in vivo contacting of a nativekidney with a bioactive cell formulation described herein together withproducts secreted from one or more enriched renal cell populations,and/or an admixture or construct containing the same.

The step of contacting a native kidney in vivo with secreted productsmay be accomplished through the use/administration of a formulationcontaining a population of secreted products from cell culture media,e.g., conditioned media, or by implantation of an enriched cellpopulation, and admixture, or a construct capable of secreting theproducts in vivo. The step of in vivo contacting provides a regenerativeeffect to the native kidney.

A variety of means for administering cells and/or secreted products tosubjects will, in view of this specification, be apparent to those ofskill in the art. Such methods include injection of the cells into atarget site in a subject.

Cells and/or secreted products can be inserted into a delivery device orvehicle, which facilitates introduction by injection or implantationinto the subjects. In certain embodiments, the delivery vehicle caninclude natural materials. In certain other embodiments, the deliveryvehicle can include synthetic materials. In one embodiment, the deliveryvehicle provides a structure to mimic or appropriately fit into theorgan's architecture. In other embodiments, the delivery vehicle isfluid-like in nature. Such delivery devices can include tubes, e.g.,catheters, for injecting cells and fluids into the body of a recipientsubject. In a preferred embodiment, the tubes additionally have aneedle, e.g., a syringe, through which the cells can be introduced intothe subject at a desired location. In some embodiments, mammaliankidney-derived cell populations are formulated for administration into ablood vessel via a catheter (where the term “catheter” is intended toinclude any of the various tube-like systems for delivery of substancesto a blood vessel). Alternatively, the cells can be inserted into oronto a biomaterial or scaffold, including but not limited to textiles,such as weaves, knits, braids, meshes, and non-wovens, perforated films,sponges and foams, and beads, such as solid or porous beads,microparticles, nanoparticles, and the like (e.g., Cultispher-S gelatinbeads-Sigma). The cells can be prepared for delivery in a variety ofdifferent forms. For example, the cells can be suspended in a solutionor gel. Cells can be mixed with a pharmaceutically acceptable carrier ordiluent in which the cells remain viable. Pharmaceutically acceptablecarriers and diluents include saline, aqueous buffer solutions, solventsand/or dispersion media. The use of such carriers and diluents is wellknown in the art. The solution is preferably sterile and fluid, and willoften be isotonic. Preferably, the solution is stable under theconditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungithrough the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. One of skill in the art willappreciate that the delivery vehicle used in the delivery of the cellpopulations and admixtures thereof can include combinations of theabove-mentioned characteristics.

Modes of administration of the formulations containing isolated renalcell population(s), for example, the B2 cell population alone or admixedwith B4′ and/or B3, include, but are not limited to, systemic,intra-renal (e.g., parenchymal), intravenous or intra-arterial injectionand injection directly into the tissue at the intended site of activity.Additional modes of administration to be used include single or multipleinjection(s) via direct laparotomy, via direct laparoscopy,transabdominal, or percutaneous. Still yet additional modes ofadministration to be used include, for example, retrograde andureteropelvic infusion. Surgical means of administration includeone-step procedures such as, but not limited to, partial nephrectomy andconstruct implantation, partial nephrectomy, partial pyelectomy,vascularization with omentum±peritoneum, multifocal biopsy needletracks, cone or pyramidal, to cylinder, and renal pole-like replacement,as well as two-step procedures including, for example, organoid-internalbioreactor for replanting. In one embodiment, the formulationscontaining admixtures of cells are delivered via the same route at thesame time. In another embodiment, each of the cell compositionscomprising the controlled admixture are delivered separately to specificlocations or via specific methodologies, either simultaneously or in atemporally-controlled manner, by one or more of the methods describedherein.

The appropriate cell implantation dosage in humans can be determinedfrom existing information relating to either the activity of the cells,for example EPO production, or extrapolated from dosing studiesconducted in preclinical studies. From in vitro culture and in vivoanimal experiments, the amount of cells can be quantified and used incalculating an appropriate dosage of implanted material. Additionally,the patient can be monitored to determine if additional implantation canbe made or implanted material reduced accordingly.

One or more other components can be added to the cell populations andadmixtures thereof, including selected extracellular matrix components,such as one or more types of collagen or hyaluronic acid known in theart, and/or growth factors, platelet-rich plasma and drugs.

Those of ordinary skill in the art will appreciate the variousformulations and methods of administration suitable for the secretedproducts described herein.

10. Articles of Manufacture and Kits

The instant disclosure further includes kits comprising the polymericmatrices and scaffolds as disclosed herein and related materials, and/orcell culture media and instructions for use. The instructions for usemay contain, for example, instructions for culture of the cells oradministration of the cells and/or cell products. In one embodiment, thepresent disclosure provides a kit comprising a scaffold as describedherein and instructions. In yet another embodiment, the kit includes anagent for detection of marker expression, reagents for use of the agent,and instructions for use. This kit may be used for the purpose ofdetermining the regenerative prognosis of a native kidney in a subjectfollowing the implantation or administration of a cell population, anadmixture, or a construct described herein. The kit may also be used todetermine the biotherapeutic efficacy of a cell population, admixture,or construct described herein.

Another embodiment is an article of manufacture containing bioactivecells useful for treatment of subjects in need. The article ofmanufacture comprises a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is effective for treating a condition and may have asterile access port (for example the container may be a solution bag ora vial having a stopper pierceable by an injection needle). At least oneactive agent in the formulation is a bioactive cell population asprovided for herein. The label or package insert indicates that theformulation is used for treating the particular condition. The label orpackage insert will further comprise instructions for administering theformulation to the patient. Articles of manufacture and kits comprisingcombinatorial therapies described herein are also contemplated. Packageinsert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In oneembodiment, the package insert indicates that the formulation is usedfor treating a disease or disorder, such as, for example, a kidneydisease or disorder. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes. Kits are also provided thatare useful for various purposes, e.g., for assessment of regenerativeoutcome. Kits can be provided which contain detection agents forurine-derived vesicles and/or their contents, e.g., nucleic acids (suchas miRNA), vesicles, exosomes, etc., as described herein. Detectionagents include, without limitation, nucleic acid primers and probes, aswell as antibodies for in vitro detection of the desired target. As withthe article of manufacture, the kit comprises a container and a label orpackage insert on or associated with the container. The container holdsa composition comprising at least one detection agent. Additionalcontainers may be included that contain, e.g., diluents and buffers orcontrol detection agents. The label or package insert may provide adescription of the composition as well as instructions for the intendedin vitro, prognostic, or diagnostic use.

11. Reports

The methods of this disclosure, when practiced for commercial purposesgenerally produce a report or summary of the regenerative prognosis. Themethods of this disclosure will produce a report comprising a predictionof the probable course or outcome of regeneration before and after anyadministration or implantation of a formulation containing a cellpopulation, an admixture, or a construct described herein. The reportmay include information on any indicator pertinent to the prognosis. Themethods and reports of this disclosure can further include storing thereport in a database. Alternatively, the method can further create arecord in a database for the subject and populate the record with data.In one embodiment the report is a paper report, in another embodimentthe report is an auditory report, in another embodiment the report is anelectronic record. It is contemplated that the report is provided to aphysician and/or the patient. The receiving of the report can furtherinclude establishing a network connection to a server computer thatincludes the data and report and requesting the data and report from theserver computer. The methods provided for herein may also be automatedin whole or in part.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. Thus, for an embodiment of the invention using one of the terms,the invention also includes another embodiment wherein one of theseterms is replaced with another of these terms. In each embodiment, theterms have their established meaning. Thus, for example, one embodimentmay encompass a formulation “comprising” a number of components, anotherembodiment would encompass a formulation “consisting essentially of” thesame components, and a third embodiment would encompass a formulation“consisting of” the same components. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The followingExamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

All patents, patent applications, and literature references cited in thepresent specification are hereby incorporated by reference in theirentirety.

EXAMPLES Example 1 Preparation of Solutions

This example provides the compositions of the various media formulationsand solutions used in the following Examples for the isolation andcharacterization of the heterogeneous renal cell population, andmanufacture of the regenerative therapy product.

TABLE 1.1 Culture Media and Solutions Material Composition TissueTransport Medium Viaspan ™ or HypoThermosol-FRS ® or DMEM Kanamycin: 100μg/mL Renal Cell Growth Medium DMEM:KSFM (50:50) 5% FBS GrowthSupplements: HGF: 10 mg/L EGF: 2.5 μg/L Insulin: 10.0 mg/L, Transferrin:5.5 mg/L Selenium: 670 μg/L Kanamycin: 100 μg/mL Tissue Wash SolutionDMEM Kanamycin: 100 μg/mL Digestion Solution Collagenase IV: 300 UnitsDispase: 5 mg/mL Calcium Chloride: 5 mM Cell Dissociation SolutionTrypLE Density Gradient Solution 7% OptiPrep OptiMEM CryopreservationSolution DMEM or HypoThermosol ® FRS 10% DMSO 10% FBS

Dulbecco's Phosphate Buffered Saline (DPBS) was used for all cellwashes.

Example 2 Isolation of the Heterogeneous Unfractionated Renal CellPopulation

This example illustrates the isolation of an unfractionated (UNFX)heterogeneous renal cell population from human. Initial tissuedissociation was performed to generate heterogeneous cell suspensionsfrom human kidney tissue.

Renal tissue via kidney biopsy provided the source material for aheterogeneous renal cell population. Renal tissue comprising one or moreof cortical, corticomedullary junction or medullary tissue may be used.It is preferred that the corticomedullary junction tissue is used.Multiple biopsy cores (minimum 2), avoiding scar tissue, were requiredfrom a CKD kidney. Renal tissue was obtained by the clinicalinvestigator from the patient at the clinical site approximately 4 weeksin advance of planned implantation of the final NKA. The tissue wastransported in the Tissue Transport Medium of Example 1.

The tissue was then washed with Tissue Wash Solution of Example 1 inorder to reduce incoming bioburden before processing the tissue for cellextractions.

Renal tissue was minced, weighed, and dissociated in the DigestionSolution of Example 1. The resulting cell suspension was neutralized inDulbecco's Modified Eagle Medium (D-MEM)+10% fetal bovine serum (FBS)(Invitrogen, Carlsbad Calif.), washed, and resuspended in serum-free,supplement-free, Keratinocyte Media (KSFM) (Invitrogen). Cellsuspensions were then subjected to a 15% (w/v) iodixanol (OptiPrep™,Sigma) gradient to remove red blood cells and debris prior to initiationof culture onto tissue culture treated polystyrene flasks or dishes at adensity of 25,000 cells per cm² in Renal Cell Growth Medium ofExample 1. For example, cells may be plated onto T500 Nunc flask at25×10⁶ cells/flask in 150 ml of 50:50 media.

Example 3 Cell Expansion of the Isolated Renal Cell Population

Renal cell expansion is dependent on the amount of tissue received andon the success of isolating renal cells from the incoming tissue.Isolated cells can be cryopreserved, if required (see infra). Renal cellgrowth kinetics may vary from sample to sample due to the inherentvariability of cells isolated from individual patients.

A defined cell expansion process was developed that accommodates therange of cell recoveries resulting from the variability of incomingtissue Table 3.1. Expansion of renal cells involves serial passages inclosed culture vessels (e.g., T-flasks, Cell Factories, HyperStacks®) inRenal Cell Growth Medium Table 1.1 using defined cell cultureprocedures.

A BPE-free medium was developed for human clinical trials to eliminatethe inherent risks associated with the use of BPE. Cell growth,phenotype (CK18) and cell function (GGT and LAP enzymatic activity) wereevaluated in BPE-free medium and compared to BPE containing medium usedin the animal studies. Renal cell growth, phenotype and function wereequivalent in the two media. (data no shown)

TABLE 3.1 Cell Recovery from Human Kidney Biopsies Renal cells (cells/10mg tissue) Source Passage 0 Passage 1 Human Kidney Tissue Samples1.51-5.36 × 106 2.40-7.48 × 107 (n = 6)

Once cell growth was observed in the initial T-flasks (passage 0) andthere were no visual signs of contamination, culture medium was replacedand changed thereafter every 2-4 days FIG. 2B. Cells were assessed toverify renal cell morphology by visual observation of cultures under themicroscope. Cultures characteristically demonstrated a tight pavement orcobblestone appearance, due to the cells clustering together. Thesemorphological characteristics vary during expansion and may not bepresent at every passage. Cell culture confluence was estimated using anImage Library of cells at various levels of confluence in the culturevessels employed throughout cell expansions.

Renal cells were passaged by trypsinization when culture vessels are atleast 50% confluent FIG. 2B. Detached cells were collected into vesselscontaining Renal Cell Growth Medium, counted and cell viabilitycalculated. At each cell passage, cells were seeded at 500-4000cells/cm2 in a sufficient number of culture vessels in order to expandthe cell number to that required for formulation of NKA FIG. 2B. Culturevessels were placed in a 37° C. incubator in a 5% CO2 environment. Asdescribed above, cell morphology and confluence was monitored and tissueculture media was replaced every 2-4 days. Table 3.2 lists the viabilityof human renal cells observed during cell isolation and expansion of sixkidney biopsies from human donors.

TABLE 3.2 Cell Viability of Human Renal Cells in Culture Passage CellViability Range (n = 6) (Average %) (%) P0 88 84-93 P1 91 80-98 P2 9492-99 P3 98 97-99

Inherent variability of tissue from different patients resulted indifferent cell yield in culture. Therefore, it is not practical tostrictly define the timing of cell passages or number and type ofculture vessels required at each passage to attain target cell numbers.Typically renal cells undergo 2 or 3 passages; however, duration ofculture and cell yield can vary depending on the cell growth rate. Thisis exemplified in FIG. 3 where the culture duration and cell yields(calculation) from 6 patients are shown.

Cells were detached for harvest or passage with 0.25% Trypsin with EDTA(Invitrogen). Viability was assessed via Trypan Blue exclusion andenumeration was performed manually using a hemacytometer or using theautomated Cellometer® counting system (Nexcelom Bioscience, LawrenceMass.).

Example 4 Cryopreservation of Cultured Cells

Expanded renal cells were routinely cryopreserved to accommodate forinherent variability of cell growth from individual patients and todeliver product on a pre-determined clinical schedule (FIG. 2B).Cryopreserved cells also provide a backup source of cells in the eventthat another NKA is needed (e.g., delay due to patient sickness,unforeseen process events, etc.). Conditions were established that havebeen used to cryopreserve cells and recover viable, functional cellsupon thawing.

For cryopreservation, cells were suspended to a final concentration ofabout 50×10⁶ cells/mL in Cryopreservation Solution (see Example 1) anddispensed into vials. One ml vials containing about 50×10⁶ cells/mL wereplaced in the freezing chamber of a controlled rate freezer and frozenat a pre-programmed rate. After freezing, the cells were transferred toa liquid nitrogen freezer for in-process storage.

Example 5 Preparation of SRC Cell Population

Selected Renal Cells (SRC) can be prepared from the final culturevessels that are grown from cryopreserved cells or directly fromexpansion cultures depending on scheduling FIG. 2B.

If using cryopreserved cells, the cells were thawed and plated on tissueculture vessels for one final expansion step. When the final culturevessels were approximately 50-100% confluent cells were ready forprocessing for SRC separation. Media exchanges and final washes of NKAdilute any residual Cryopreservation Solution in the final product.

Once the final cell culture vessels have reached at least 50% confluencethe culture vessels were transferred to a hypoxic incubator set for 2%oxygen in a 5% CO2 environment at 37° C. and cultured overnight. SeeFIG. 2C. Cells may be held in the oxygen-controlled incubator set to 2%oxygen for as long as 48 hours. Exposure to the more physiologicallyrelevant low-oxygen (2%) environment improved cell separation efficiencyand enabled greater detection of hypoxia-induced markers such as VEGF.

After the cells have been exposed to the hypoxic conditions for asufficient time (e.g., overnight to 48 hours), the cells were detachedwith 0.25% Trypsin with EDTA (Invitrogen). Viability was assessed viaTrypan Blue exclusion and enumeration was performed manually using ahemacytometer or using the automated Cellometer® counting system(Nexcelom Bioscience, Lawrence Mass.). Cells were washed once with DPBSand resuspended to about 850×10⁶ cells/mL in DPBS.

Density gradient centrifugation was used to separate harvested renalcell populations based on cell buoyant density. Renal cell suspensionswere separated on single-step 7% iodixanol Density Gradient Solution(OptiPrep; 60% (w/v) in OptiMEM; see Example 1).

The 7% OptiPrep gradient solution was prepared and refractive indexindicative of desired density was measured (R.I. 1.3456+/−0.0004) priorto use. Harvested renal cells were layered on top of the gradientsolution. The density gradient was centrifuged at 800 g for 20 min atroom temperature (without brake) in either centrifuge tubes or a cellprocessor (e.g., COBE 2991). The cellular fraction exhibiting buoyantdensity greater than approximately 1.045 g/mL was collected aftercentrifugation as a distinct pellet FIG. 4. Cells maintaining a buoyantdensity of less than 1.045 g/mL were excluded and discarded.

The SRC pellet was re-suspended in DPBS (FIG. 2C). The carry-over ofresidual OptiPrep, FBS, culture medium and ancillary materials in thefinal product is minimized by 4 DPBS wash and 1 Gelatin Solution steps.

Example 6 Cells with Therapeutic Potential can be Isolated andPropagated from Normal and Chronically-Diseased Kidney Tissue

The objective of the present study was to determine the functionalcharacterization of human NKA cells through high content analysis (HCA).High-content imaging (HCl) provides simultaneous imaging of multiplesub-cellular events using two or more fluorescent probes (multiplexing)across a number of samples. High-content Analysis (HCA) providessimultaneous quantitative measurement of multiple cellular parameterscaptured in High-Content Images. In brief, unfractionated (UNFX)cultures were generated (Aboushwareb et al., World J Urol 26, 295, 2008)and maintained independently from core biopsies taken from five humankidneys with advanced chronic kidney disease (CKD) and three non-CKDhuman kidneys using standard biopsy procedures. After (2) passages ofUNFX ex vivo, cells were harvested and subjected to density gradientmethods (as described in Example 2 of Basu et al., WO 2012/064369) togenerate subfractions, including subfractions B2, B3, and/or B4.

Human kidney tissues were procured from non-CKD and CKD human donors assummarized in Table 10.1 of Ilagan et al. PCT/US2011/036347. FIG. 4 ofIlagan et al. PCT/US2011/036347 shows histopathologic features of theHK17 and HK19 samples. Ex vivo cultures were established from allnon-CKD (3/3) and CKD (5/5) kidneys. High content analysis (HCA) ofalbumin transport in human NKA cells defining regions of interest (ROI)is shown in FIG. 5 (HCA of albumin transport in human NKA cells) ofIlagan et al. PCT/US2011/036347. Quantitative comparison of albumintransport in NKA cells derived from non-CKD and CKD kidney is shown inFIG. 6 of Ilagan et al. PCT/US2011/036347. As shown in FIG. 6 of Ilaganet al. PCT/US2011/036347, albumin transport is not compromised inCKD-derived NKA cultures. Comparative analysis of marker expressionbetween tubular-enriched B2 and tubular cell-depleted B4 subfractions isshown in FIG. 7 (CK8/18/19) of Ilagan et al. PCT/US2011/036347.

Comparative functional analysis of albumin transport betweentubular-enriched B2 and tubular cell-depleted B4 subfractions is shownin FIG. 8 of Ilagan et al. PCT/US2011/036347. Subfraction B2 is enrichedin proximal tubule cells and thus exhibits increased albumin-transportfunction.

Albumin uptake: Culture media of cells grown to confluency in 24-well,collagen IV plates (BD Biocoat™) was replaced for 18-24 hours withphenol red-free, serum-free, low-glucose DMEM (pr-/s-/lg DMEM)containing 1× antimycotic/antibiotic and 2 mM glutamine. Immediatelyprior to assay, cells were washed and incubated for 30 minutes withpr-/s-/Ig DMEM+10 mM HEPES, 2 mM glutamine, 1.8 mM CaCl2, and 1 mMMgCl2. Cells were exposed to 25 μg/mL rhodamine-conjugated bovinealbumin (Invitrogen) for 30 min, washed with ice cold PBS to stopendocytosis and fixed immediately with 2% paraformaldehyde containing 25μg/mL Hoechst nuclear dye. For inhibition experiments, 1 μMreceptor-associated protein (RAP) (Ray Biotech, Inc., Norcross Ga.) wasadded 10 minutes prior to albumin addition. Microscopic imaging andanalysis was performed with a BD Pathway™ 855 High-Content BioImager(Becton Dickinson) (see Kelley et al. Am J Physiol Renal Physiol. 2010November; 299(5):F1026-39. Epub Sep. 8, 2010).

In conclusion, HCA yields cellular level data and can reveal populationsdynamics that are undetectable by other assays, i.e., gene or proteinexpression. A quantifiable ex-vivo HCA assay for measuring albumintransport (HCA-AT) function can be utilized to characterize human renaltubular cells as components of human NKA prototypes. HCA-AT enabledcomparative evaluation of cellular function, showing that albumintransport-competent cells were retained in NKA cultures derived fromhuman CKD kidneys. It was also shown that specific subfractions of NKAcultures, B2 and B4, were distinct in phenotype and function, with B2representing a tubular cell-enriched fraction with enhanced albumintransport activity. The B2 cell subpopulation from human CKD arephenotypically and functionally analogous to rodent B2 cells thatdemonstrated efficacy in vivo (as shown above).

Example 7 Low-Oxygen Culture Prior to Gradient Affects BandDistribution, Composition, & Gene Expression

To determine the effect of oxygen conditions on distribution andcomposition of prototypes B2 and B4, neokidney cell preparations fromdifferent species were exposed to different oxygen conditions prior tothe gradient step. A rodent neo-kidney augmentation (NKA) cellpreparation (RK069) was established using standard procedures for ratcell isolation and culture initiation, as described in Kelley et al.,2010, supra. All flasks were cultured for 2-3 days in 21% (atmospheric)oxygen conditions. Media was changed and half of the flasks were thenrelocated to an oxygen-controlled incubator set to 2% oxygen, while theremaining flasks were kept at the 21% oxygen conditions, for anadditional 24 hours. Cells were then harvested from each set ofconditions using standard enzymatic harvesting procedures describedsupra. Step gradients were prepared according to standard procedures andthe “normoxic” (21% oxygen) and “hypoxic” (2% oxygen) cultures wereharvested separately and applied side-by-side to identical stepgradients. While 4 bands and a pellet were generated in both conditions,the distribution of the cells throughout the gradient was different in21% and 2% oxygen-cultured batches (Table 7.1). Specifically, the yieldof B2 was increased with hypoxia, with a concomitant decrease in 83.Furthermore, the expression of B4-specific genes (such aserythropoietin) was enhanced in the resulting gradient generated fromthe hypoxic-cultured cells (FIG. 73 of Presnell et al. WO/2010/056328).

A canine NKA cell preparation (DK008) was established using standardprocedures for dog cell isolation and culture (analogous to rodentisolation and culture procedures), as described in Basu et al., WO2012/064369. All flasks were cultured for 4 days in 21% (atmospheric)oxygen conditions, then a subset of flasks were transferred to hypoxia(2%) for 24 hours while a subset of the flasks were maintained at 21%.Subsequently, each set of flasks was harvested and subjected toidentical step gradients. Similar to the rat results, thehypoxic-cultured dog cells distributed throughout the gradientdifferently than the atmospheric oxygen-cultured dog cells (Table 7.1).Again, the yield of 82 was increased with hypoxic exposure prior togradient, along with a concomitant decrease in distribution into 83.

TABLE 7.1 Rat (RK069) Dog (DK008) 2% O2 21% O2 2% O2 21% O2 B1 0.77%0.24% 1.20% 0.70% B2 88.50% 79.90% 64.80% 36.70% B3 10.50% 19.80% 29.10%40.20% B4 0.23% 0.17% 4.40% 21.90%

The above data show that pre-gradient exposure to hypoxia enhancescomposition of B2 as well as the distribution of specific specializedcells (erythropoietin-producing cells, vascular cells, and glomerularcells) into 84. Thus, hypoxic culture, followed by density-gradientseparation as described in Basu et al., supra, is an effective way togenerate ‘B2’ and ‘B4’ cell populations, across species.

Example 8 Characterization of an Unfractionated Mixture of Renal CellsIsolated from an Autoimmune Glomerulonephritis Patient Sample

An unfractionated mixture of renal cells was isolated, as describedabove, from an autoimmune glomerulonephritis patient sample. Todetermine the unbiased genotypic composition of specific subpopulationsof renal cells isolated and expanded from kidney tissue, quantitativereal time PCR (qRTPCR) analysis (Brunskill et al., Dev. Cell. 2008November; 15(5):781-791) was employed to identify differentialcell-type-specific and pathway-specific gene expression patterns amongthe cell subfractions. As shown in Table 6.1 of Ilagan et al.PCT/US2011/036347, HK20 is an autoimmune glomerulonephritis patientsample. As shown in Table 6.2 of Ilagan et al. PCT/US2011/036347, cellsgenerated from HK20 are lacking glomerular cells, as determined byqRTPCR.

Example 9 Genetic Profiling of Therapeutically Relevant Renal BioactiveCell Populations Isolated from a Case of Focal SegmentalGlomerulosclerosis

To determine the unbiased genotypic composition of specificsubpopulations of renal cells isolated and expanded from kidney tissue,quantitative real time PCR (qRTPCR) analysis (Brunskill et al., supra2008) was employed to identify differential cell-type-specific andpathway-specific gene expression patterns among the cell subfractions.Human preparation HK023, derived from a case of focal segmentalglomerulosclerosis (FSGS) in which a large portion of glomeruli had beendestroyed, was evaluated for presence of glomerular cells in the B4fraction at the time of harvest. In brief, unfractionated (UNFX)cultures were generated (Aboushwareb et al., World J Urol 26, 295, 2008)and maintained independently from each of (4) core biopsies taken fromthe kidney using standard biopsy procedures. After (2) passages of UNFXex vivo, cells were harvested and subjected to density gradient methodsaccording to Example 6 of Basu et al., WO2012/064369 to generatesubfractions, including subfraction B4, which is known to be enrichedfor endocrine, vascular, and glomerular cells based on work conducted inrodent, dog, and other human specimens.

The B4 fractions were collected separately from each independent UNFXsample of HK023, appearing as distinct bands of cells with buoyantdensity between 1.063-1.091 g/mL. RNA was isolated from each sample andexamined for expression of Podocin (glomerular cell marker) and PECAM(endothelial cell marker) by quantitative real-time PCR. As expectedfrom a biopsy-generated sample from a case of severe FSGS, the presenceof podocin(+) glomerular cells in B4 fractions was inconsistent, withpodocin undetectable in 2/4 of the samples. In contrast, PECAM+ vascularcells were consistently present in the B4 fractions of 4/4 of thebiopsy-initiated cultures. Thus, the B4 fraction can be isolated at the1.063-1.091 g/mL density range, even from human kidneys with severedisease states.

TABLE 9.1 Expression of Podocin and PECAM for detection of glomerularand vascular cells in subfraction B4 isolated from a case of FSGS.HK023/Biopsy RQ (Podocin)/B4 RQ (PECAM)/84 #1/p2 0.188 0.003 #2/p2 ND0.02 #3/p2 40.1 0.001 #4/p2 ND 0.003

Further, as shown in Table 7.2 of Ilagan et al. PCT/US2011/036347, humansample (HK018) displayed undetected Podocin (glomerular marker) byqRTPCR after density gradient centrifugation.

Example 10 Enrichment/Depletion of Viable Kidney Cell Types UsingFluorescent Activated Cell Sorting (FACS)

One or more isolated kidney cells may be enriched, and/or one or morespecific kidney cell types may be depleted from isolated primary kidneytissue using fluorescent activated cell sorting (FACS).

Reagents:

70% ethanol; Wash buffer (PBS); 50:50 Kidney cell medium (50% DMEM highglucose): 50% Keratinocyte-SFM; Trypan Blue 0.4%; Primary antibodies totarget kidney cell population such as CD31 for kidney endothelial cellsand Nephrin for kidney glomerular cells. Matched isotype specificfluorescent secondary antibodies; Staining buffer (0.05% BSA in PBS).

Procedure:

Following standard procedures for cleaning the biological safety cabinet(BSC), a single cell suspension of kidney cells from either primaryisolation or cultured cells may be obtained from a T500 T/C treatedflask and resuspend in kidney cell medium and place on ice. Cell countand viability is then determined using trypan blue exclusion method. Forkidney cell enrichment/depletion of, for example, glomerular cells orendothelial cells from a heterogeneous population, between 10 and 50×10⁶live cells with a viability of at least 70% are obtained. Theheterogeneous population of kidney cells is then stained with primaryantibody specific for target cell type at a starting concentration of 1g/0.1 ml of staining buffer/1×10⁶ cells (titer if necessary). Targetantibody can be conjugated such as CD31 PE (specific for kidneyendothelial cells) or un-conjugated such as Nephrin (specific for kidneyglomerular cells).

Cells are then stained for 30 minutes on ice or at 4° C. protected fromlight. After 30 minutes of incubation, cells are washed bycentrifugation at 300×g for 5 min. The pellet is then resuspended ineither PBS or staining buffer depending on whether a conjugated isotypespecific secondary antibody is required. If cells are labeled with afluorochrome conjugated primary antibody, cells are resuspended in 2 mlsof PBS per 10⁷ cells and proceed to FACS aria or equivalent cell sorter.If cells are not labeled with a fluorochrome conjugated antibody, thencells are labeled with an isotype specific fluorochrome conjugatedsecondary antibody at a starting concentration of 1 ug/0.1 ml/10⁶ cells.

Cells are then stained for 30 min. on ice or at 4° C. protected fromlight. After 30 minutes of incubation, cells are washed bycentrifugation at 300×g for 5 min. After centrifugation, the pellet isresuspended in PBS at a concentration of 5×10⁶/ml of PBS and then 4 mlsper 12×75 mm is transferred to a sterile tube.

FACs Aria is prepared for live cell sterile sorting per manufacturer'sinstructions (BD FACs Aria User Manual). The sample tube is loaded intothe FACs Aria and PMT voltages are adjusted after acquisition begins.The gates are drawn to select kidney specific cells types usingfluorescent intensity using a specific wavelength. Another gate is drawnto select the negative population. Once the desired gates have beendrawn to encapsulate the positive target population and the negativepopulation, the cells are sorted using manufacturer's instructions.

The positive target population is collected in one 15 ml conical tubeand the negative population in another 15 ml conical tube filled with 1ml of kidney cell medium. After collection, a sample from each tube isanalyzed by flow cytometry to determine purity. Collected cells arewashed by centrifugation at 300×g for 5 min. and the pellet isresuspended in kidney cell medium for further analysis andexperimentation.

Example 11 Enrichment/Depletion of Kidney Cell Types Using Magnetic CellSorting

One or more isolated kidney cells may be enriched and/or one or morespecific kidney cell types may be depleted from isolated primary kidneytissue.

Reagents:

70% ethanol, Wash buffer (PBS), 50:50 Kidney cell medium (50% DMEM highglucose): 50% Keratinocyte-SFM, Trypan Blue 0.4%, Running Buffer (PBS, 2mM EDTA, 0.5% BSA), Rinsing Buffer (PBS, 2 mM EDTA), Cleaning Solution(70% v/v ethanol), Miltenyi FCR Blocking reagent, Miltenyi microbeadsspecific for either IgG isotype, target antibody such as CD31(PECAM) orNephrin, or secondary antibody.

Procedure:

Following standard procedures for cleaning the biological safety cabinet(BSC), a single cell suspension of kidney cells from either primaryisolation or culture is obtained and resuspended in kidney cell medium.Cell count and viability is determined using trypan blue exclusionmethod. For kidney cell enrichment/depletion of, for example, glomerularcells or endothelial cells from a heterogeneous population, at least 10⁶up to 4×10⁹ live cells with a viability of at least 70% is obtained.

The best separation for enrichment/depletion approach is determinedbased on target cell of interest. For enrichment of a target frequencyof less than 10%, for example, glomerular cells using Nephrin antibody,the Miltenyi autoMACS, or equivalent, instrument program POSSELDS(double positive selection in sensitive mode) is used. For depletion ofa target frequency of greater than 10%, the Miltenyi autoMACS, orequivalent, instrument program DEPLETES (depletion in sensitive mode) isused.

Live cells are labeled with target specific primary antibody, forexample, Nephrin rb polyclonal antibody for glomerular cells, by adding1 μg/10⁶ cells/0.1 ml of PBS with 0.05% BSA in a 15 ml conicalcentrifuge tube, followed by incubation for 15 minutes at 4° C.

After labeling, cells are washed to remove unbound primary antibody byadding 1-2 ml of buffer per 10⁷ cells followed by centrifugation at300×g for 5 min. After washing, isotype specific secondary antibody,such as chicken anti-rabbit PE at 1 ug/10⁶/0.1 ml of PBS with 0.05% BSA,is added, followed by incubation for 15 minutes at 4° C.

After incubation, cells are washed to remove unbound secondary antibodyby adding 1-2 ml of buffer per 10⁷ cells followed by centrifugation at300×g for 5 min. The supernatant is removed, and the cell pellet isresuspended in 60 μl of buffer per 10⁷ total cells followed by additionof 20 μl of FCR blocking reagent per 10⁷ total cells, which is thenmixed well.

Add 20 μl of direct MACS microbeads (such as anti-PE microbeads) and mixand then incubate for 15 min at 4° C.

After incubation, cells are washed by adding 10-20× the labeling volumeof buffer and centrifuging the cell suspension at 300×g for 5 min. andresuspending the cell pellet in 500 μl-2 mls of buffer per 10⁸ cells.

Per manufacturer's instructions, the autoMACS system is cleaned andprimed in preparation for magnetic cell separation using autoMACS. Newsterile collection tubes are placed under the outlet ports. The autoMACScell separation program is chosen. For selection the POSSELDS program ischosen. For depletion the DEPLETES program is chosen.

The labeled cells are inserted at uptake port, then beginning theprogram. After cell selection or depletion, samples are collected andplaced on ice until use. Purity of the depleted or selected sample isverified by flow cytometry.

Example 12 Phenotypic Characterization of the Enriched HeterogeneousRenal Cell Population

The following example details the use of flow cytometry to characterizethe selected heterogeneous human renal cells of Example 5. Theheterogeneous renal cell population was composed primarily of renalepithelial cells that are well known for their regenerative potential.Other parenchymal (vascular) and stromal (collecting duct) cells may besparsely present in the autologous cell population.

Cell phenotype is monitored by expression analysis of renal cell markersusing flow cytometry. Phenotypic analysis of cells is based on the useof antigenic markers specific for the cell type being analyzed. Flowcytometric analysis provides a quantitative measure of cells in thesample population which express the antigenic marker being analyzed.

A variety of markers have been reported in the literature as beinguseful for phenotypic characterization of renal cells: (i) cytokeratins;(ii) transport membrane proteins (aquaporins and cubilin); (iii) cellbinding molecules (adherins, cluster of differentiation, and lectins);and (iv) metabolic enzymes (glutathione). Since the majority of cellsfound in cultures derived from whole kidney digests are epithelial andendothelial cells, the markers examined focus on the expression ofproteins specific for these two groups.

Cytokeratins are a family of intermediate filament proteins expressed bymany types of epithelial cells to varying degrees. The subset ofcytokeratins expressed by an epithelial cell depends upon the type ofepithelium. For example, cytokeratins 7, 8, 18 and 19 are all expressedby normal simple epithelia of the kidney and remaining urogenital tractas well as the digestive and respiratory tracts. These cytokeratins incombination are responsible for the structural integrity of epithelialcells. This combination represents both the acidic (type I) and basic(type II) keratin families and is found abundantly expressed in renalcells (Oosterwijk et al., J Histochem Cytochem, 38(3):385-392, 1990).Preferred cytokeratins for use herein are CK8, CK18, CK19 andcombinations thereof

Aquaporins are transport membrane proteins which allow the passage ofwater into and out of the cell, while preventing the passage of ions andother solutes. There are thirteen aquaporins described in theliterature, with six of these being found in the kidney (Nielsen et al.,J Histochem Cytochem, 38(3):385-392, 2002). Aquaporin2, by exertingtight control in regulating water flow, is responsible for the plasmamembranes of renal collecting duct epithelial cells having a highpermeability to water, thus permitting water to flow in the direction ofan osmotic gradient (Bedford et al., J Am Soc Nephrol, 14(10):2581-2587,2003; Takata et al., Histochem Cell Biol, 130(2):197-209, 2008; Tamma etal., Endocrinology, 148(3):1118-1130, 2007). Aquaporin1 ischaracteristic of the proximal tubules (Baer et al., Cells TissuesOrgans:184(1), 16-22, 2006; Nielsen et al., 2002, supra).

Cubilin is a transport membrane receptor protein. When it co-localizeswith the protein megalin, together they promote the internalization ofcubilin-bound ligands such as albumin. Cubilin is located within theepithelium of the intestine and the kidney (Christensen, Am J PhysiolRenal Physiol, 280(4):F562-573, 2001).

CXCR4 is a transport membrane protein which serves as a chemokinereceptor for SDF1. Upon ligand binding, intracellular calcium levelsincrease and MAPK1/MAPK3 activation is increased. CXCR4 isconstitutively expressed in the kidney and plays an important role inkidney development and tubulogenesis (Ueland et al., Dev Dyn,238(5):1083-1091, 2009).

Cadherins are calcium-dependent cell adhesion proteins. They areclassified into four groups, with the E-cadherins being found inepithelial tissue, and are involved in regulating mobility andproliferation. E-cadherin is a transmembrane glycoprotein which has beenfound to be localized in the adherins Junctions of epithelial cellswhich make up the distal tubules in the kidney (Prozialeck et al., BMCPhysiol, 4:10, 2004; Shen et al., Mod Pathol, 18(7):933-940, 2005).

DBA (Dolichos biflorus agglutinin) is an α-N-acetylgalactosamine-bindinglectin (cell binding protein) carried on the surface of renal collectingduct structures, and is regarded and used as a general marker ofdeveloping renal collecting ducts and distal tubules (Michael et al., JAnat 210(1):89-97, 2007; Lazzeri et al., J Am Soc Nephrol 18(12):3128-3138, 2007).

Cluster of differentiation 31 (CD31; also known as platelet endothelialcell adhesion molecule, PECAM-1) is a cell adhesion protein which isexpressed by select populations of immune cells as well as endothelialcells. In endothelial cells, this protein is concentrated at the cellborders (DeLisser, 1997). Cluster of differentiation 146 (CD146) isinvolved in cell adhesion and cohesion of endothelial cells atintercellular junctions associated with the actin cytoskeleton. Stronglyexpressed by blood vessel endothelium and smooth muscle, CD146 iscurrently used as a marker for endothelial cell lineage (Malyszko etal., J Clin Endocrinol Metab, 89(9):4620-4627, 2004), and is the canineequivalent of CD31.

Gamma-glutamyl transpeptidase (GGT) is a metabolic enzyme that catalyzesthe transfer of the gamma-glutamyl moiety of glutathione to an acceptorthat may be an amino acid, a peptide, or water, to form glutamate. Thisenzyme also plays a role in the synthesis and degradation of glutathioneand the transfer of amino acids across the cell membrane. GGT is presentin the cell membranes of many tissues, including the proximal tubulecells of kidneys (Horiuchi et al., Eur J Biochem, 87(3):429-437, 1978;Pretlow et al., J Histochem Cytochem, 35(4):483-487, 1987; Welbourne etal., Am J Physiol, 277(4 Pt 2):F501-505, 1999). Table 12.1 provides alist of the specific types of renal cells expressing these markers asdetected by flow cytometry.

TABLE 12.1 Phenotypic Markers for SRC Characterization Antigenic MarkerReactivity CK8/18/19 Epithelial cells, proximal and distal tubules CK8Epithelial cells, proximal tubules CK18 Epithelial cells, proximaltubules CK19 Epithelial cells, collecting ducts, distal tubules CK7Epithelial cells, collecting ducts, distal tubules CKCR4 Epithelialcells, proximal and distal tubules E-cadherin Epithelial cells, distaltubules Cubilin Epithelial cells, proximal tubules Aquaporin 1Epithelial cells, proximal tubules, descending thin limb GGT1 Fetal andadult kidney cells, proximal tubules Aquaporin2 Renal collecting ductcells, distal tubules DBA Renal collecting duct cells, distal tubulesCD31 Endothelial cells of the kidney (rat) CD146 Endothelial cells ofthe kidney (canine, human)

The SRC cells of Example 5 were investigated for phenotype of specificbiomarkers.

For immunophenotyping: The specific antibody was added to 100microliters of cell suspension and the mixture was incubated in the darkfor 30-45 minutes at 4° C. After incubation, cells were washed with PBSand centrifuged to remove excess antibody. Cells were resuspended tocells/microliters PBS and analyzed by flow cytometry. Flow cytometryanalysis was performed with a FACSAria flow cytometer (Becton Dickinson)and FlowJo software (Treestar, Inc.). Antibodies used to characterizethe surface marker phenotype are shown in Tables 12.2 and 12.3.Isotype-specific primary antibody negative controls were used in allexperiments. Appropriate isotype-matched controls were used to gatenegative populations.

TABLE 12.2 Phenotype Panel for Human SRC in NKA Reported in INDAntigenic Kidney Cell Commercial Marker Distribution ComponentDescription Primary Phenotypic Profile CK18 Epithelial cells,Cytoplasmic C-04 clone, Ms mAb proximal tubules IgG1, Abcam ab668 GGT1Proximal tubules Membrane Ms mAb IgG2a, Abcam ab55138 AQP2 Distaltubules, Membrane Rb pAb IgG, Abcam collecting ducts ab62628 ExpandedPhenotypic Panel CK8, 18, 19 Proximal and Cytoplasmic Ms mAb IgG1, Abcamdistal tubules ab41825 CK8 Proximal tubules Cytoplasmic Ms mAb IgG1,Abcam ab9023 CK19 Distal tubules, Cytoplasmic Clone 8A-17, Ms mAbcollecting ducts Abcam ab7755 CK7 Distal tubules, Cytoplasmic Ms mAbIgG1, Abcam collecting ducts ab41825 CXCR4 Proximal and Membrane Clone12G5, Ms mAb (Fusin) distal tubules IgG2a, R&D Systems MAB170 E-cadherinDistal tubules Membrane Ms mAb IgG2a, BD (CD324) 610182 Cubilin Proximaltubules Membrane Gt pAb IgG, Santa Cruz Biotechnology, sc- 23644 AQP1Proximal tubules, Membrane Clone 1/22, Ms mAb descending thin IgG2a,Abcam ab9566 limb DBA Distal tubules, Membrane Agglutinin, Vectorcollecting ducts Labs B-1035 CD31 Endothelial cells Membrane Ms mAbIgG1, BD (PECAM-1) (rat) 555444 CD146 Endothelial cells Membrane CloneP1H12, Ms (human, dog) mAb IgG1, Abcam ab24577 Abbreviations: Gt, goat;Ms, mouse; mAb, monoclonal antibody; pAb, polyclonal antibody; Rb,rabbit.

TABLE 12.3 Additional Phenotypic Markers for Evaluation of Human SRCAntigenic Kidney Marker Distribution Commercial Description AQP4Collecting ducts Clone 8J301, Ms mAb IgG1, US Biological A3000-14B CD24Glomerulus and Rb pAb IgG, Abcam ab110448 proximal tubules(progenitor/stem cell marker) CD54 Glomerulus Clone HA58, Ms mAb IgG1,(ICAM-1) (endothelial BD 559771 cells, human) CD73 Renal Cortex Gt pAbIgG, Santa Cruz (interstitial Biotechnology, sc-14684 fibroblasts) CD117Proximal tubules Clone 104D2, Ms mAb 104D2, (stem cell marker) BD 340867CD133 Glomerulus and Rb pAb IgG, Abcam ab19898 proximal tubules(progenitor/stem cell marker) CK8, 18 Proximal and Ms mAb IgG2a, Abcamdistal tubules ab15224 CK40 to 67 Proximal tubules Clone AE1/AE3, Ms mAbIgG1, Dako M3515 N-cadherin Proximal tubules Ms mAb IgG1, BD 610921Pan-cadherin Proximal tubules Clone CH-19, Ms mAb IgG1, Abcam ab6528Calbindin Distal tubules Ms mAb IgG1, US Biological C0113-15 CalponinGlomerulus Ms mAb IgG1, Dako M3556 (interstitial fibroblasts) Connexin43 Glomerulus Clone 4EG.2, Ms mAb IgG1, Abcam ab79010 EPO Renal CortexMs mAb IgG2a, US Biological (erythropoeitin) (interstitial E3455-13fibroblasts) GLEPP1 Glomerulus Gt pAb IgG, Santa Cruz (glomerularBiotechnology sc-33415 epithelial protein 1)

 GST-1 Proximal Rb pAb IgG, Santa Cruz (alpha tubules Biotechnologysc-459 glutathione S-transferase) Haptoglobulin Glomerulus Chicken IgY,US Biological 03-003-02 Itgb1 Collecting Clone JB1B, Ms mAb IgG2a,(Integrin; 

 1) ducts Abcam ab30388 KIM-1/TIM-1 Proximal Clone 212211, Ms mAb IgG2b,(kidney injury tubules R&D Systems MAB1750 molecule-1/ T-cellimmunoglobulin and mucin- containing molecule) Megalin Proximal and RbpAb IgG, Santa Cruz distal tubules Biotechnology, sc-25470 MAP-2Proximal and Clone M13, Ms mAb IgG1, (microtubule- distal tubulesZymed/Life Technologies associated 13-1500 protein 2) Nephrin GlomerulusMs mAb IgG1, US Biological N2028-50D NKCC Distal tubules Rb pAb IgG,Santa Cruz (Na—K—Cl- Biotechnology sc-133823 cotransporters) OAT-1Proximal Rb pAb IgG, US Biological (organic anion tubules 041836transporter 1) Osteopontin Proximal and Clone 53, Ms mAb IgG2a, distaltubules Abcam 69498 PCLP1 Glomerulus Gt pAb IgG, Santa Cruz podocalyxin-Biotechnology sc-10503 like 1 molecule) Podocin Glomerulus Rb pAb IgG,Santa Cruz Biotechnology sc-21009 SMA Glomerulus Ms mAb IgG2a, DakoM0851 (smooth muscle (interstitial alpha-actin) fibroblasts)Synaptopodin Glomerulus Rb pAb IgG, Santa Cruz Biotechnology sc-50459THP Distal tubules Ms mAb IgG2a, Santa Cruz (tamm-horsfall Biotechnologysc-20631 protein) Vimentin Proximal tubules Rb pAb IgG, Atlas(progenitor/stem Antibodies HPA001762 cell marker)

Cell suspensions were generated from initial tissue dissociation ortrypsinization of adherent cultures and analyzed by flow cytometry toidentify cellular components. Antibodies employed are listed in Tables12.2 and 12.3 (above). Isotype-specific primary antibody negativecontrols were used in all experiments. Labeled cells were analyzed witha FACSAria flow cytometer (Becton Dickinson) and FlowJo software(Treestar, Inc.). Appropriate isotype-matched controls were used to gatenegative populations. After an overnight incubation at 4° C., the cellswere pelleted, washed twice with Triton Buffer (0.2% Triton X-100 inPBS), resuspended in 1 mL of DBPS containing secondary antibody goatanti-mouse IgG2A conjugated to the fluorochrome Alexa A647 (Invitrogen),and incubated for an additional 30 minutes. Cells were then washed andresuspended in 1 mL of PBS for analysis as per manufacturer instructionsusing FACSAria and FlowJo software. As a negative control, cells wereincubated in parallel with isotype-matched monoclonal antibodiesconjugated to the same fluorochrome.

FIG. 5 (Phenotype Distribution) shows quantified expression of thesemarkers in SRC populations plotted as percentage values of eachphenotype in the population.

CK8/18/19 are the most consistently expressed renal cell proteinsdetected across species. GGT1 and Aquaporin-1 (AQP1) are expressedconsistently but at varying levels. DBA, Aquaporin2 (AQP2), E-cadherin(CAD), CK7, and CXCR4 are also observed at modest levels though withmore variability, and CD31/146 and Cubilin were lowest in expression.Table 12.4 provides the selected markers, range and mean percentagevalues of phenotypic in SRC and the rationale for their selection.

TABLE 12.4 Marker Selected for Phenotypic Analysis of SRC PhenotypicExpression Expression Marker Range Average Rationale level CK18 81.1 to99.7% 96.7% Epithelial High (n = 87) marker GGT1  4.5 to 81.2% 50.7%Functional Moderate (n = 63) Tubular marker AQP2  3.0 to 53.7% 26.8%Collecting Low* (n = 24) duct marker *Collecting duct epithelial cellsare expected to be low in SRC based on their buoyant density

SRC Gene Expression

The gene expression profile of SRC isolated from human renal cellcultures by quantitative real-time polymerase chain reaction (qPCR),including those of aquaporin2, E-cadherin, cubulin, VEGF and CD31 thatwere also tested for protein production. Genotypic markers in Table 12.5are representative of cell populations that might be expected to befound in the renal cell cultures. NCAD, Cubilin and CYP2R1 are markersof tubular epithelial cells, AQP2 and ECAD are markers of collectingduct and distal tubules. Podocin and Nephrin are markers of podocytes.VEGF and CD31 are endothelial markers. VEGF and EPO are oxygenresponsive genes with related mRNA present in a variety of differenttissue and cell types.

Gene probes used were obtained from TaqMan. Passage 2 human renal cellswere harvested at 70-90% confluence. RNA was purified from the cellsusing Qiagen's RNeasy Plus Mini Kit following the protocol forPurification of Total RNA from Animal Cells. cDNA was generated from avolume of RNA equal to 1.4 μg using Invitrogen's SuperScript® VILO™ cDNASynthesis Kit following the manufacturer's instructions. Averaged qPCRdata for SRC populations (n=3) is shown in Table 12.5.

The results suggest that a population of tubular epithelial cells ispresent as evidenced by relatively higher level of expression of NCAD,Cubilin and CYP2R1. Distal Collecting Duct Tubule and Distal Tubulemarkers AQP2 and ECAD are relatively low and CD31, an endothelial markeris even lower (Table 12.5).

TABLE 12.5 Gene Expression Analysis of Human SRC Human Gene Average StdGene Name Designation Marker RQ Error Aquaporin2 AQP2 Distal 0.201 0.201Tubule, Collecting Duct E-cadherin/ ECAD/ Distal 0.318 0.191 Cadherin 1,CDH1 Tubule Type 1 Neuronal NCAD/ Proximal 3.027 0.208 Cadherin/ CDH2Tubule Cadherin 2, Type 1 Cubilin CUBN Tubular 4.319 1.036 Nephrin NPHS1Podocyte 0.768 0.422 Podocin NPHS2 Podocyte 0.000 0.000 ErythropoietinEPO Cortical 2.795 0.426 Fibroblast Vitamin D CYP2R1 Tubular 1.562 0.02824-Hydroxylase Vascular VEGFA Endothelial 2.232 0.121 Endothelial GrowthFactor A Platelet/ PECAM1/ Endothelial 0.005 0.005 Endothelial CD31 CellAdhesion Molecule/CD31

Phenotypic and functional markers have been chosen based upon earlygenotypic evaluation. VEGF gene expression levels were high andaquaporin2 gene expression levels were low which is consistent with theprotein analysis data (Table 12.4 and Table 12.6).

SRC Enzymatic Activity

Presence of viable cells and SRC function was demonstrated by metabolismof PrestoBlue and production of VEGF and KIM-1.

SRC actively secrete proteins that can be detected through analysis ofconditioned medium. Cell function is assessed by the ability of cells tometabolize PrestoBlue and secrete VEGF (Vascular Endothelial GrowthFactor) and KIM-1 (Kidney Injury Molecule-1).

Viable functioning cells can be monitored in NKA by their ability tometabolize PrestoBlue. PrestoBlue Cell Viability Reagent is a modifiedresazurin-based assay reagent that is a cell permeable, non-fluorescentblue dye. Upon entry into cells which are sufficiently viable toproliferate, the dye is reduced, via natural cell processes involvingdehydrogenase enzymes, to a bright red fluorophore that can be measuredby fluorescence or absorbance.

Biomolecules VEGF and KIM-1 represent a selection of molecules fromthose proposed as sensitive and specific analytical nonclinicalbiomarkers of kidney injury and function (Sistare, 2010; Warnock, 2010).In vivo, both of these markers are indicative of tubular function,injury and/or repair and in vitro are recognized features of tubularepithelial cell cultures. KIM-1 is an extracellular protein anchored inthe membrane of renal proximal tubule cells that serves to recognize andphagocytose apoptotic cells which are shed during injury and cellturnover. VEGF, constitutively expressed by kidney cells, is a pivotalangiogenic and pro-survival factor that promotes cell division,migration, endothelial cell survival and vascular sprouting. SRC havebeen characterized as constitutively expressing VEGF mRNA (Table 12.5)and actively produce the protein (Table 12.6). These proteins may bedetected in culture medium exposed to renal cells and SRC. Table 12.6presents VEGF and KIM-1 quantities present in conditioned medium fromrenal cells and SRC cultures. Renal cells were cultured to nearconfluence. Conditioned medium from overnight exposure to the renal cellcultures and SRC was tested for VEGF and KIM-1.

TABLE 12.6 Production of VEGF and KIM-1 by Human Renal Cells and SRCCondi- VEGF KIM-1 tioned ng/million ng/million Medium ng/mL cells ng/mLcells Renal Cell 0.50 to 2.42 2.98 to 14.6 0.20 to 3.41 1.14 to 15.2Culture (n = 15) SRC 0.80 to 3.85  4.83 to 23.07 0.32 to 2.10  1.93 to12.59 (n = 14)

Cell function of SRC, pre-formulation, was also evaluated by measuringthe activity of two specific enzymes; GGT (γ-glutamyl transpeptidase)and LAP (leucine aminopeptidase) (Chung 1982, J Cell Biol95(1):118-126), found in kidney proximal tubules. Methods to measure theactivity of these enzymes in cells utilize an enzyme-specific substratein solution that, when added to cells expressing active enzyme, arecleaved, releasing a chromogenic product (Nachlas, 1960 J BiophysBiochem Cytol 7:261-264; Tate, 1974 Proc Natl Acad Sci USA71(9):3329-3333). The absorbance of the cell-exposed solution ismeasured and is relative to the amount of cleavage product resultingfrom active enzyme. The substrate utilized for GGT is L-glutamic acidγ-p-nitroanalide hydrochloride and for LAP is L-leucine p-nitroanalide.FIG. 6 shows LAP and GGT activity in six SRC samples produced from humandonors (BP1-BP4).

Summary of SRC Characterization

Cell morphology was monitored during cell expansion by comparison ofculture observations with images in an Image Library. Cell growthkinetics were monitored at each cell passage. Cell growth is expected tobe variable from patient to patient. SRC counts and viability weremonitored by Trypan Blue dye exclusion and/or metabolism of PrestoBlue.SRC are characterized by phenotypic expression of CK18, GGT1. Inaddition AQP2 expression may be monitored. Metabolism of PrestoBlue andproduction of VEGF and KIM-1 are used as markers for the presence ofviable and functional SRC. SRC function can be further elucidated withgene expression profiling and measurement of enzymatic activity with LAPand GGT.

Example 13 Biomaterial Preparation

The Biomaterial used in NKA (Gelatin Solution) is characterized via twokey parameters:

Concentration—

Concentration of Gelatin Solution is measured by absorbance at 280 nmusing a spectrophotometer. The gelatin concentration is determined froma calibration curve of absorbance versus concentration.

Inversion Test—

The inversion test provides a visual assessment of the ability of theGelatin Solution to form and maintain a gel at a temperature of 2-8° C.and for the gel to liquefy at room temperature.

Elucidation of Other Biomaterial Characteristics

Biomaterials used in NKA can be further characterized for therheological properties and viscosity. Rheology and viscosity testingwill be performed for verification purposes only and are intended to beused for expanded characterization of biomaterials obtained from othervendors.

Rheological properties of the Biomaterial can be measured first at 4°C., then at 25° C. through the use of a Couette Cell style rheometer.The sample is equilibrated for at least 30 minutes at each temperature.An acceptable storage modulus (G′>10) at the lower temperature reflectsthe ability of the solution to form and maintain a gel at NKA shippingand transport temperature of 2-8° C. An acceptable loss modulus (G″<10)at the higher temperature reflects the ability of the gel to liquefy atroom temperature as required for delivery and implantation of NKA.

Viscosity of the Biomaterial is measured using a cone and plateviscometer at 37° C. and a shear rate of 200-300 s−1. Solutions withviscosities in range of 1.05-1.35 cP can be efficiently deliveredthrough 18-27 gauge needles.

In preparation for NKA formulation, gelatin is dissolved in DPBS to aspecified concentration (0.88%±0.12% w/v) to form a Gelatin Solution(FIG. 2D). Gelatin Solution was filter sterilized through a 0.1 μmfilter and aliquoted into tubes. Samples were taken for release of theGelatin Solution prior to freezing or formulation of NKA. The gelledhydrogel is stored refrigerated or frozen as a bulk material ready forformulation (FIG. 2D).

Example 14 NKA Formulation

Washed SRC from Example 5 were counted using Trypan Blue dye exclusion.Gelatin Solution was removed from cold storage and liquefied by warmingto 26-30° C. A volume of SRC suspension containing the required numberof cells was centrifuged and re-suspended in liquefied Gelatin Solutionfor a final wash step. This suspension was centrifuged and the SRCpellet is re-suspended in sufficient Gelatin Solution to achieve aresultant SRC concentration of 100×10⁶ cells/mL in the formulated NKA(FIG. 2D).

NKA is presented in a sterile, single-use 10 mL syringe. The finalvolume was calculated from the concentration of 100×10⁶ SRC/mL of NKAand the target dose of 3.0×10⁶ SRC/g kidney weight (estimated by MRI).

Example 15 NKA Filling and Gelation

NKA product was aseptically filled into the syringe in the NKA packagein a BSC for tissue processing and cell culture operations (FIG. 2D).Dynamic air sampling was performed for the duration of the fillingprocess, including viable and non-viable sampling.

Formulated NKA was contained in a 50 mL sterile centrifuge tube. Asterile cannula was attached to a 10 mL transfer syringe. NKA wasmanually drawn into the transfer syringe from the 50 mL tube via thecannula. The cannula was removed and the transfer syringe was connectedto the luer-lock fitting at the end of the NKA package tubing. NKA wastransferred to the syringe in the NKA package by depressing the plungeron the transfer syringe. A minimum of 8.5 mL of product was transferredto the syringe in the NKA package. Air entrapped in the syringe wasremoved by inverting the syringe and slowly depressing the plunger.After filling was complete, the tubing on the NKA package was sealedwith an RF Sealer. Remaining product in the transfer syringe wasreturned to the 50 mL tube. Quality control (release testing) sampleswere taken from the 50 mL tube. The NKA package was rotated for aminimum of 2 hours to keep the cells in suspension while cooling to 2-8°C. to form the final gelled NKA (FIG. 2D).

Rapid cooling was required for gelation to take place so that cells donot settle in the Gelatin Solution. The temperature of the GelatinSolution in a syringe was monitored as it was placed into refrigeratedconditions. Rapid temperature drop was observed. After 1 hour, thetemperature had dropped to within 0.3° C. of the final temperature 4.4°C.

Cooling of the Gelatin Solution starts the gelation process but a finiteamount to time was required for the formed gel to stabilize such thatthe SRC will remain suspended in the gel on storage. Syringes containingformulated NKA were rotated either overnight or for 1.25 hours and thenheld upright overnight. Subsequently, the contents were removed and cellconcentration was measured in four different segments of the product.Analysis indicates that there was no difference among the four segments,thus no measurable cell settling occurs once NKA has rotated at coldtemperature for a minimum of 1.25 hours (data not shown).

Example 16 NKA Packaging and Shipping

NKA was packaged along with the appropriate documentation into the NKAShipper. The Shipper is designed to maintain a temperature range of 2-8°C. during transportation to the clinical site. Cooled (gelled)formulated product has a shelf life of 3 days.

A temperature recorder was included with the NKA package to monitor thetemperature during shipment. The Batch Number of NKA was verifiedagainst the unique Patient ID recorded in the shipping/receiving logbook by the Quality group. NKA Shipper was shipped to the clinical sitevia courier or similar secure transportation.

Example 17 Implantation of the NKA (SRC Cell Population)

This example demonstrates the regenerative properties of the selectedheterogeneous human renal cells.

NKA Delivery System

NKA delivery system was composed of a cannula (needle) compatible withcell delivery and a syringe. Different vendors use the terms cannula orneedle to describe cell delivery products. For this description the termcannula and needle are used interchangeably. The proposed clinical trialutilized the same delivery system (cannula and syringe) used in theanimal studies adapted to human size and anatomy. A laparoscopicsurgical procedure was used.

The main component of NKA delivery system was the cannula. A cannulathat was compatible with NKA was used.

NKA Implantation

In preparation for implantation, NKA was allowed to warm to roomtemperature just before injection into the kidney to liquefy theproduct.

NKA was targeted for injection into the kidney cortex via a needle orcannula and syringe compatible with cell delivery. The use of a piercingneedle (cannula) to penetrate the kidney capsule allowed introduction ofthe delivery needle/cannula into the kidney cortex. The syringecontaining NKA was attached to the delivery needle and NKA was injectedinto multiple sites into the kidney cortex. The schematic in FIG. 7illustrates the concept of injecting NKA into a kidney using a needlecompatible with cell delivery and distribution into a solid organ.

NKA was delivered directly into the kidney cortex. NKA delivery inpatients used a standardized laparoscopic procedure.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of identifying a heterogeneous renalcell population, said method comprising the steps: isolating cells froma mammalian kidney sample; exposing said isolated cells to labeleddetection moieties for biomarkers, wherein each labeled detection moietyis directed to a different biomarker and is labeled, wherein thebiomarkers comprise GGT-1 and a cytokeratin; determining the percentageof cells that express each of said different biomarker; and determiningwhether the cell population comprises cells that express VEGF and KIM-1.2. The method of claim 1, wherein the biomarkers further comprise AQP1,AQP2, AQP4, Calbindin, Calponin, CD117, CD133, CD146, CD24, CD31(PECAM-1), CD54 (ICAM-1), CD73 Connexin 43, Cubilin, CXCR4 (Fusin), DBA,E-cadherin (CD324), EPO (erythropoeitin), GLEPP1 (glomerular epithelialprotein 1), Haptoglobin, Itgb1 (Integrin β1), MAP-2(microtubule-associated protein 2), Megalin, N-cadherin, Nephrin, NKCC(Na—K—Cl-cotransporters), OAT-1 (organic anion transporter 1),Osteopontin, Pan-cadherin, PCLP1 (podocalyxin-like 1 molecule), Podocin,SMA (smooth muscle alpha-actin), Synaptopodin, THP (tamm-horsfallprotein), Vimentin-, αGST-1 (alpha glutathione 5-transferase glutathioneS-transferase), or any combination thereof.
 3. The method of claim 1,wherein each labeled detection moiety is an antibody.
 4. The method ofclaim 1, wherein isolating the cells comprises establishing an in vitroculture of the cells, wherein the mammalian kidney sample is a renaltissue biopsy or whole kidney tissue.
 5. The method according to claim4, wherein determining whether the cell population comprises cells thatexpress VEGF and KIM-1 comprises (i) obtaining a supernatant from aculture of the heterogeneous renal cell population, and (ii) detectingthe level of VEGF and KIM-1 in the supernatant.
 6. The method of claim1, further comprising identifying the heterogeneous renal cellpopulation as suitable for implantation and/or eliciting a regenerativeresponse if (i) greater than 18% of the cells express GGT-1; and (ii)the cell population comprises cells that express VEGF and KIM-1.
 7. Amethod of treating kidney disease in a mammal comprising administeringto the mammal an effective amount of cells from a heterogeneous renalcell population identified as suitable for implantation and/or elicitinga regenerative response according to claim
 6. 8. The method of claim 1,further comprising identifying the heterogeneous renal cell populationas suitable for implantation and/or eliciting a regenerative response if(i) greater than 18% of the cells within the cell population expressGGT-1 and greater than 80% of the cells within the cell populationexpress a cytokeratin; and (ii) the cell population comprises cells thatexpress VEGF and KIM-1.
 9. The method of claim 8, wherein thecytokeratin is CK18.
 10. A method of treating kidney disease in a mammalcomprising administering to the mammal an effective amount of cells froma heterogeneous renal cell population identified as suitable forimplantation and/or eliciting a regenerative response according to claim8.
 11. The method according to claim 10, wherein the cells are culturedon a matrix.
 12. The method according to claim 10, wherein the cells aresuspended in a gelatin-based biomaterial.
 13. The method according toclaim 10, wherein the mammal is human.
 14. The method of claim 1,wherein the biomarkers further comprise AQP2, and the method furthercomprises identifying the heterogeneous renal cell population assuitable for implantation and/or eliciting a regenerative response if(i) 4.5% to 81.2% of the cells in the cell population express GGT-1,3.0% to 53.7% of the cells within the cell population express AQP2, and81.1% to 99.7% of the cells within the cell population express CK18; and(ii) the cell population comprises cells that express VEGF and KIM-1.